U.S. patent application number 12/968970 was filed with the patent office on 2011-11-03 for holographic image projection method and holographic image projection system.
This patent application is currently assigned to OLYMPUS CORPORATION. Invention is credited to Yoshiaki Murayama.
Application Number | 20110267663 12/968970 |
Document ID | / |
Family ID | 44858056 |
Filed Date | 2011-11-03 |
United States Patent
Application |
20110267663 |
Kind Code |
A1 |
Murayama; Yoshiaki |
November 3, 2011 |
HOLOGRAPHIC IMAGE PROJECTION METHOD AND HOLOGRAPHIC IMAGE
PROJECTION SYSTEM
Abstract
Provided is a hologram-image projection method including a step
of setting three-dimensional position information in a specimen for
a plurality of focal points where laser light is to be focused via
an objective lens; a step of performing reverse ray tracing from
the individual focal points to an entrance pupil position of the
objective lens by using the position information set for the
individual focal points in the specimen, a refractive index of the
specimen, and overall characteristic data of the objective lens, to
calculate a wavefront of the laser light at the entrance pupil
position from the individual focal points; a step of calculating a
combined wavefront by combining the plurality of calculated
wavefronts; a step of setting a phase pattern to be applied to a
wavefront modulating device on the basis of the calculated combined
wavefront; and a step of applying the set phase pattern to the
wavefront modulating device, causing the laser light to be incident
thereon, and focusing the laser light whose wavefront is modulated
by the phase pattern on the specimen via the objective lens.
Inventors: |
Murayama; Yoshiaki; (Tokyo,
JP) |
Assignee: |
OLYMPUS CORPORATION
Tokyo
JP
|
Family ID: |
44858056 |
Appl. No.: |
12/968970 |
Filed: |
December 15, 2010 |
Current U.S.
Class: |
359/9 |
Current CPC
Class: |
G03H 2001/2213 20130101;
G03H 2225/32 20130101; G03H 1/2294 20130101; G03H 1/2249 20130101;
G03H 2210/30 20130101; G03H 1/08 20130101 |
Class at
Publication: |
359/9 |
International
Class: |
G03H 1/08 20060101
G03H001/08 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 21, 2009 |
JP |
2009-289883 |
Dec 21, 2009 |
JP |
2009-289884 |
Dec 22, 2009 |
JP |
2009-291503 |
Claims
1. A hologram-image projection method comprising: a position
setting step of setting three-dimensional position information in a
specimen for a plurality of focal points where laser light is to be
focused via an objective lens; a wavefront calculating step of
performing reverse ray tracing from the individual focal points
assumed at set positions to an entrance pupil position of the
objective lens by using the position information set for the
individual focal points in the specimen, a refractive index of the
specimen, and overall characteristic data of the objective lens, to
calculate wavefronts of the laser light at the entrance pupil
position from the individual focal points; a wavefront combining
step of calculating a combined wavefront by combining the plurality
of wavefronts calculated for the individual focal points; a
phase-pattern setting step of setting a phase pattern to be applied
to a wavefront modulating device on the basis of the calculated
combined wavefront; and a focusing step of applying the set phase
pattern to the wavefront modulating device, causing the laser light
to be incident thereon, and focusing the laser light whose
wavefront is modulated by the phase pattern on the specimen via the
objective lens.
2. A hologram-image projection method according to claim 1, wherein
the wavefront combining step is a step of calculating the combined
wavefront by calculating a linear summation of the plurality of
wavefronts.
3. A hologram-image projection method according to claim 1, wherein
the wavefront combining step is a step of calculating the combined
wavefront by arraying respective divided regions of the plurality
of wavefronts with a ratio equal to the reciprocal of the number of
focal points.
4. A hologram-image projection method comprising: a position
setting step of setting three-dimensional position information in a
specimen for a plurality of focal points where laser light is to be
focused via an objective lens; a virtual-image creating step of
creating a virtual image in which the individual focal points are
projected on a prescribed reference plane in the specimen using the
set position information of the individual focal points in the
specimen; a wavefront calculating step of calculating a wavefront
of the laser light at an entrance pupil position of the objective
lens by Fourier transforming the created virtual image using the
refractive index of the specimen and overall characteristic data of
the objective lens; a phase-pattern setting step of setting a phase
pattern to be applied to a wavefront modulating device on the basis
of the calculated wavefront; and a focusing step of applying the
set phase pattern to the wavefront modulating device, causing the
laser light to be incident thereon, and focusing the laser light
whose wavefront is modulated by the phase pattern on the specimen
via the objective lens.
5. A hologram-image projection method according to claim 4,
wherein: the position setting step includes a plane position
setting step of setting plane position information in the specimen
for the plurality of focal points, and a depth-information setting
step of setting depth information relative to the reference plane
for the individual focal points; the virtual-image creating step
includes a first-virtual-image creating step of creating a first
virtual image for when it is assumed that the individual focal
points are focused on a prescribed reference plane in the specimen
to form a plurality of spots, by using the plane position
information in the specimen for the individual focal points, and a
second-virtual-image creating step of creating a second virtual
image by correcting a corresponding spot in the first virtual image
on the basis of the set depth information of the individual focal
points.
6. A hologram-image projection method according to claim 5, wherein
the second-virtual-image creating step is a step of correcting the
corresponding spot in the first virtual image by substituting the
spot in the first virtual image with a spots of the laser light
obtained by ray tracing the laser light focused at the individual
focal points up to the reference plane.
7. A hologram-image projection method according to claim 5, wherein
the second-virtual-image creating step includes a step in which,
with the objective lens disposed so that a focal position of the
objective lens is displaced from the reference plane by a
prescribed distance in the optical axis direction, a coefficient of
proportionality is calculated by dividing a change in vector
information, occurring when vector information representing
aberrations at a focal plane of the objective lens varied, by the
prescribed distance so that light is focused at the reference
plane, and a step of correcting the spots using vector information
obtained by multiplying the coefficient of proportionality and the
depth information for each spot.
8. A hologram-image projecting apparatus comprising: a wavefront
modulating device that modulates a wavefront of laser light with a
phase pattern; an objective lens that focuses the laser light
modulated by the wavefront modulating device on a specimen; a
position-information setting section for setting three-dimensional
position information in the specimen for a plurality of focal
points where the laser light is to be focused via the objective
lens; a wavefront calculating section that performs reverse ray
tracing from the individual focal points assumed at set positions
to an entrance pupil position of the objective lens by using the
position information, inside the specimen, set for the individual
focal points by the position-information setting section, a
refractive index of the specimen, and overall characteristic data
of the objective lens, to calculate wavefronts of the laser light
at the entrance pupil position from the individual focal points; a
wavefront combining section that calculates a combined wavefront by
combining the plurality of wavefronts calculated by the wavefront
calculating section for the individual focal points; and a
phase-pattern setting section that sets the phase pattern to be
applied to the wavefront modulating device on the basis of the
calculated combined wavefront.
9. A hologram-image projecting apparatus comprising: a wavefront
modulating device that modulates a wavefront of laser light with a
phase pattern; an objective lens that focuses the laser light
modulated by the wavefront modulating device on a specimen; a
position-information setting section for setting three-dimensional
position information, inside the specimen, for a plurality of focal
points where the laser light is to be focused via the objective
lens; a virtual-image creating section of creating a virtual image
in which the individual focal points are projected on a prescribed
reference plane in the specimen by using the position information,
inside the specimen, set for the individual focal points by the
position-information setting section; a wavefront calculating
section that calculates a wavefront of the laser light at an
entrance pupil position of the objective lens by Fourier
transforming the virtual image created by the virtual-image
creating section using a refractive index of the specimen and
overall characteristic data of the objective lens; and a
phase-pattern setting section that sets the phase pattern to be
applied to the wavefront modulating device on the basis of the
calculated wavefront.
10. A hologram-image projection method comprising: a position
setting step of setting, for a plurality of focal points where
laser light is to be focused via an objective lens, position
information on a focal plane of the objective lens in the specimen;
a wavefront calculating step of performing reverse ray tracing from
the individual focal points assumed at set positions to an entrance
pupil position of the objective lens by using the position
information, in the specimen, set for the individual focal points,
a refractive index of the specimen, and lens data of individual
lenses constituting the objective lens, to calculate wavefronts of
the laser light at the entrance pupil position from the individual
focal points; a wavefront combining step of calculating a combined
wavefront by combining the plurality of wavefronts calculated for
the individual focal points; a phase-pattern setting step of
setting a phase pattern to be applied to a wavefront modulating
device on the basis of the calculated combined wavefront; and a
focusing step of applying the set phase pattern to the wavefront
modulating device, causing the laser light to be incident thereon,
and focusing the laser light whose wavefront is modulated by the
phase pattern on the specimen via the objective lens.
11. A hologram-image projection method according to claim 10,
wherein the wavefront combining step is a step of calculating the
combined wavefront by calculating a linear summation of the
plurality of wavefronts.
12. A hologram-image projection method according to claim 10,
wherein the wavefront combining step is a step of calculating the
combined wavefront by arraying respective divided regions of the
plurality of wavefronts with a ratio equal to the reciprocal of the
number of focal points.
13. A hologram-image projection method comprising: a position
setting step of setting, for a plurality of focal points where
laser light is to be focused via an objective lens, position
information on a focal plane of the objective lens in the specimen;
a virtual-image creating step of creating a virtual image having a
plurality of spots formed at the individual focal points, by
performing forward ray tracing from an entrance pupil position of
the objective lens to the focal plane using the position
information, in the specimen, set for the individual focal points,
a refractive index of the specimen, and lens data of individual
lenses constituting the objective lens; a wavefront calculating
step of calculating the wavefront so that, by varying the wavefront
of the laser light at the entrance pupil position of the objective
lens, the diameters of the individual spots in the created virtual
image are minimized; a phase-pattern setting step of setting a
phase pattern to be applied to a wavefront modulating device on the
basis of the calculated wavefront; and a focusing step of applying
the set phase pattern to the wavefront modulating device, causing
the laser light to be incident thereon, and focusing the laser
light whose wavefront is modulated by the phase pattern on the
specimen via the objective lens.
14. A hologram-image projection method according to claim 13,
wherein the position information is a focal-point map including the
plurality of focal points, which are assumed to be on the focal
plane of the objective lens in the specimen; the virtual-image
creating step includes a step of calculating the wavefront of the
laser light at the entrance pupil position of the objective lens by
Fourier transforming the focal-point map, and a step of creating
the virtual image by performing forward ray tracing from the
entrance pupil position of the objective lens to the focal plane,
for the laser light having the calculated wavefront, by using the
refractive index of the specimen and the lens data of the
individual lenses constituting the objective lens.
15. A hologram-image projecting apparatus comprising: a wavefront
modulating device that modulates a wavefront of laser light with a
phase pattern; an objective lens that focuses the laser light
modulated by the wavefront modulating device on a specimen; a
position-information setting section for setting, for a plurality
of focal points on a focal plane where laser light is to be focused
via an objective lens, position information on the focal plane of
the objective lens in the specimen; a wavefront calculating section
that performs reverse ray tracing from the individual focal points
assumed at the set positions to an entrance pupil position of the
objective lens by using the position information of the individual
focal points set by the position-information setting section, a
refractive index of the specimen, and lens data of individual
lenses constituting the objective lens to calculate wavefronts of
the laser light at the entrance pupil position from the individual
focal points; a wavefront combining section that calculates a
combined wavefront by combining the plurality of wavefronts
calculated for the individual focal points; and a phase-pattern
setting section that sets the phase pattern to be applied to the
wavefront modulating device on the basis of the calculated combined
wavefront.
16. A hologram-image projection apparatus comprising: a laser light
source; a wavefront modulating device that modulates a wavefront of
laser light emitted from the laser light source with a phase
pattern; an objective lens that focuses the laser light on a
specimen; an incident-angle adjusting section that adjusts an
incident angle of the laser light at the entrance pupil position of
the objective lens; a focal-position setting section that sets the
positions of a plurality of focal points where the laser light is
to be focused in the specimen; a wavefront measuring section that
measures wavefronts, at the entrance pupil position of the
objective lens, or a position optically conjugate therewith, for
return light returning from the individual focal points at the
positions set by the focal-position setting section; a wavefront
combining section that calculates a combined wavefront by combining
the plurality of wavefronts of the return light measured by the
wavefront measuring section; and a phase-pattern setting section
that sets the phase pattern to be applied to the wavefront
modulating device on the basis of the calculated combined wavefront
and outputs the phase pattern to the wavefront modulating
device.
17. A hologram-image projection apparatus according to claim 16,
wherein the wavefront combining section calculates the combined
wavefront by calculating a linear summation of the plurality of
return light wavefronts measured by the wavefront measuring
section.
18. A hologram-image projection apparatus according to claim 16,
wherein the wavefront combining section calculates the combined
wavefront by arraying respective divided regions of the plurality
of return light wavefronts measured by the wavefront measuring
section with a ratio equal to the reciprocal of the number of focal
points.
19. A hologram-image projection apparatus according to claim 16,
wherein the wavefront measuring section includes a splitting
portion that generates reference light by splitting the laser
light; a combining section that combines the reference light and
the return light from the focal point; and an interference-light
detector that detects interference light of the return light and
the reference light combined by the combining section.
20. A hologram-image projection apparatus according to claim 16,
wherein the wavefront measuring section includes a pinhole member
disposed at a position that is optically conjugate with the focal
position of the objective lens; and a Hartmann sensor that detects
the return light from the focal point, which has passed through the
pinhole member.
Description
TECHNICAL FIELD
[0001] The present invention relates to a hologram-image projection
method and a hologram-image projection apparatus.
[0002] This application is based on Japanese Patent Applications
No. 2009-289883, No. 2009-289884, and No. 2009-291503, the contents
of which are incorporated herein by reference.
BACKGROUND ART
[0003] In the related art, use of a hologram is known for
simultaneously radiating light at a plurality of locations on a
specimen (for example, see NPL 1).
[0004] In this method, a fluorescence image of the specimen is
acquired with a fluorescence microscope or the like, a stimulus
light irradiation pattern is created by identifying a plurality of
sites of interest in the acquired fluorescence image where a light
stimulus etc. is to be applied, and this irradiation pattern is
Fourier transformed, thereby creating a hologram phase pattern.
Then, by applying the created phase pattern to a wavefront
modulating device and causing a substantially collimated beam of
laser light guided from a light source to be incident on this
wavefront modulating device, the laser light is modulated, and the
modulated laser light is focused by an objective lens. Accordingly,
a hologram image is projected on the specimen, so that the stimulus
light is focused at a plurality of locations simultaneously.
CITATION LIST
Non Patent Literature
{NPL 1}
[0005] Volodymyr Nikolenko et al, "SLM microscopy:scanless
two-photon imaging and photostimulation with spatial light
modulators", Frontiers in Neural Circuits, Vol 2, Article 5, 19
Dec. 2008, p 1-15
SUMMARY OF INVENTION
Technical Problem
[0006] The present invention provides a hologram-image projection
method and a hologram-image projection apparatus in which light can
be focused simultaneously at sites of interest disposed at
different positions in the depthwise direction in a specimen.
[0007] In addition, the present invention provides a hologram-image
projection method and a hologram-image projection apparatus in
which laser light can be focused simultaneously at a plurality of
desired focal points in a specimen, even if aberrations are present
in the objective lens.
[0008] Furthermore, the present invention provides a hologram-image
projection apparatus in which laser light can be focused
simultaneously at a plurality of desired focal points in a
specimen, even when causes of various kinds of aberrations
coexist.
Solution to Problem
[0009] The present invention provides the following solutions.
[0010] A first aspect of the present invention is a hologram-image
projection method including a position setting step of setting
three-dimensional position information in a specimen for a
plurality of focal points where laser light is to be focused via an
objective lens; a wavefront calculating step of performing reverse
ray tracing from the individual focal points assumed at the set
positions to an entrance pupil position of the objective lens by
using the position information set for the individual focal points
in the specimen, a refractive index of the specimen, and overall
characteristic data of the objective lens, to calculate wavefronts
of the laser light at the entrance pupil position from the
individual focal points; a wavefront combining step of calculating
a combined wavefront by combining the plurality of wavefronts
calculated for the individual focal points; a phase-pattern setting
step of setting a phase pattern to be applied to a wavefront
modulating device on the basis of the calculated combined
wavefront; and a focusing step of applying the set phase pattern to
the wavefront modulating device, causing the laser light to be
incident thereon, and focusing the laser light whose wavefront is
modulated by the phase pattern on the specimen via the objective
lens.
[0011] A second aspect of the present invention is a hologram-image
projection method including a position setting step of setting
three-dimensional position information in a specimen for a
plurality of focal points where laser light is to be focused via an
objective lens; a virtual-image creating step of creating a virtual
image in which the individual focal points are projected on a
prescribed reference plane in the specimen using the set position
information of the individual focal points in the specimen; a
wavefront calculating step of calculating a wavefront of the laser
light at an entrance pupil position of the objective lens by
Fourier transforming the created virtual image using the refractive
index of the specimen and overall characteristic data of the
objective lens; a phase-pattern setting step of setting a phase
pattern to be applied to a wavefront modulating device on the basis
of the calculated wavefront; and a focusing step of applying the
set phase pattern to the wavefront modulating device, causing the
laser light to be incident thereon, and focusing the laser light
whose wavefront is modulated by the phase pattern on the specimen
via the objective lens.
[0012] A third aspect of the present invention is a hologram-image
projecting apparatus including a wavefront modulating device that
modulates a wavefront of laser light with a phase pattern; an
objective lens that focuses the laser light modulated by the
wavefront modulating device on a specimen; a position-information
setting section for setting three-dimensional position information
in the specimen for a plurality of focal points where the laser
light is to be focused via the objective lens; a wavefront
calculating section that performs reverse ray tracing from the
individual focal points assumed at the set positions to an entrance
pupil position of the objective lens by using the position
information, inside the specimen, set for the individual focal
points by the position-information setting section, a refractive
index of the specimen, and overall characteristic data of the
objective lens, to calculate wavefronts of the laser light at the
entrance pupil position from the individual focal points; a
wavefront combining section that calculates a combined wavefront by
combining the plurality of wavefronts calculated by the wavefront
calculating section for the individual focal points; and a
phase-pattern setting section that sets the phase pattern to be
applied to the wavefront modulating device on the basis of the
calculated combined wavefront.
[0013] A fourth aspect of the present invention is a hologram-image
projecting apparatus including a wavefront modulating device that
modulates a wavefront of laser light with a phase pattern; an
objective lens that focuses the laser light modulated by the
wavefront modulating device on a specimen; a position-information
setting section for setting three-dimensional position information,
inside the specimen, for a plurality of focal points where the
laser light is to be focused via the objective lens; a
virtual-image creating section of creating a virtual image in which
the individual focal points are projected on a prescribed reference
plane in the specimen by using the position information, inside the
specimen, set for the individual focal points by the
position-information setting section; a wavefront calculating
section that calculates a wavefront of the laser light at an
entrance pupil position of the objective lens by Fourier
transforming the virtual image created by the virtual-image
creating section using a refractive index of the specimen and
overall characteristic data of the objective lens; and a
phase-pattern setting section that sets the phase pattern to be
applied to the wavefront modulating device on the basis of the
calculated wavefront.
[0014] A fifth aspect of the present invention is a hologram-image
projection method including a position setting step of setting, for
a plurality of focal points where laser light is to be focused via
an objective lens, position information on a focal plane of the
objective lens in the specimen; a wavefront calculating step of
performing reverse ray tracing from the individual focal points
assumed at the set positions to an entrance pupil position of the
objective lens by using the position information, in the specimen,
set for the individual focal points, a refractive index of the
specimen, and lens data of individual lenses constituting the
objective lens, to calculate wavefronts of the laser light at the
entrance pupil position from the individual focal points; a
wavefront combining step of calculating a combined wavefront by
combining the plurality of wavefronts calculated for the individual
focal points; a phase-pattern setting step of setting a phase
pattern to be applied to a wavefront modulating device on the basis
of the calculated combined wavefront; and a focusing step of
applying the set phase pattern to the wavefront modulating device,
causing the laser light to be incident thereon, and focusing the
laser light whose wavefront is modulated by the phase pattern on
the specimen via the objective lens.
[0015] A sixth aspect of the present invention is a hologram-image
projection method including a position setting step of setting, for
a plurality of focal points where laser light is to be focused via
an objective lens, position information on a focal plane of the
objective lens in the specimen; a virtual-image creating step of
creating a virtual image having a plurality of spots formed at the
individual focal points, by performing forward ray tracing from an
entrance pupil position of the objective lens to the focal plane
using the position information, in the specimen, set for the
individual focal points, a refractive index of the specimen, and
lens data of individual lenses constituting the objective lens; a
wavefront calculating step of calculating the wavefront so that, by
varying the wavefront of the laser light at the entrance pupil
position of the objective lens, the diameters of the individual
spots in the created virtual image are minimized; a phase-pattern
setting step of setting a phase pattern to be applied to a
wavefront modulating device on the basis of the calculated
wavefront; and a focusing step of applying the set phase pattern to
the wavefront modulating device, causing the laser light to be
incident thereon, and focusing the laser light whose wavefront is
modulated by the phase pattern on the specimen via the objective
lens.
[0016] A seventh aspect of the present invention is a
hologram-image projecting apparatus including a wavefront
modulating device that modulates a wavefront of laser light with a
phase pattern; an objective lens that focuses the laser light
modulated by the wavefront modulating device on a specimen; a
position-information setting section for setting, for a plurality
of focal points on a focal plane where laser light is to be focused
via an objective lens, position information on the focal plane of
the objective lens in the specimen; a wavefront calculating section
that performs reverse ray tracing from the individual focal points
assumed at the set positions to an entrance pupil position of the
objective lens by using the position information of the individual
focal points set by the position-information setting section, a
refractive index of the specimen, and lens data of individual
lenses constituting the objective lens, to calculate wavefronts of
the laser light at the entrance pupil position from the individual
focal points; a wavefront combining section that calculates a
combined wavefront by combining the plurality of wavefronts
calculated for the individual focal points; and a phase-pattern
setting section that sets the phase pattern to be applied to the
wavefront modulating device on the basis of the calculated combined
wavefront.
[0017] An eighth aspect of the present invention is a
hologram-image projection apparatus including a laser light source;
a wavefront modulating device that modulates a wavefront of laser
light emitted from the laser light source with a phase pattern; an
objective lens that focuses the laser light on a specimen; an
incident-angle adjusting section that adjusts an incident angle of
the laser light at the entrance pupil position of the objective
lens; a focal-position setting section that sets the positions of a
plurality of focal points where the laser light is to be focused in
the specimen; a wavefront measuring section that measures
wavefronts, at the entrance pupil position of the objective lens,
or a position optically conjugate therewith, for return light
returning from the individual focal points at the positions set by
the focal-position setting section; a wavefront combining section
that calculates a combined wavefront by combining the plurality of
wavefronts of the return light measured by the wavefront measuring
section; and a phase-pattern setting section that sets the phase
pattern to be applied to the wavefront modulating device on the
basis of the calculated combined wavefront and outputs the phase
pattern to the wavefront modulating device.
BRIEF DESCRIPTION OF DRAWINGS
[0018] {FIG. 1} FIG. 1 is an overall configuration diagram
schematically showing a hologram-image projection apparatus
according to a first embodiment of the present invention.
[0019] {FIG. 2} FIG. 2 is a flowchart for explaining a
hologram-image projection method according to the first embodiment
of the present invention, using the hologram-image projection
apparatus in FIG. 1.
[0020] {FIG. 3} FIG. 3 is an overall configuration diagram
schematically showing a hologram-image projection apparatus
according to a second embodiment of the present invention.
[0021] {FIG. 4} FIG. 4 is a flowchart for explaining a
hologram-image projection method according to the second embodiment
of the present invention, using the hologram-image projection
apparatus in FIG. 3.
[0022] {FIG. 5A} FIG. 5A is a diagram for explaining a method of
creating a second virtual image by forward ray tracing, in the
hologram-image projection method in FIG. 4.
[0023] {FIG. 5B} FIG. 5B is a diagram for explaining a method of
creating a second virtual image by reverse ray tracing, in the
hologram-image projection method in FIG. 4.
[0024] {FIG. 6A} FIG. 6A is a diagram showing a second virtual
image created by forward ray tracing using the method in FIG.
5A.
[0025] {FIG. 6B} FIG. 6B is a diagram showing a second virtual
image created by reverse ray tracing using the method in FIG.
5B.
[0026] {FIG. 7A} FIG. 7A is a diagram for explaining a modification
of the hologram-image projection method in FIG. 4, schematically
showing a state where a reference plane is coincident with a focal
plane of an objective lens.
[0027] {FIG. 7B} FIG. 7B is a diagram for explaining a modification
of the hologram-image projection method in FIG. 4, schematically
showing a state where the focal plane of the objective lens is
shifted in the depthwise direction from the reference plane.
[0028] {FIG. 7C} FIG. 7C is a diagram for explaining a modification
of the hologram-image projection method in FIG. 4, schematically
showing a state where Zernike coefficients are adjusted to make the
focal plane of the objective lens coincident with the reference
plane.
[0029] {FIG. 8} FIG. 8 is an overall configuration diagram
schematically showing a hologram-image projection apparatus
according to a third embodiment of the present invention.
[0030] {FIG. 9} FIG. 9 is a flowchart for explaining a
hologram-image projection method according to the third embodiment
of the present invention, using the hologram-image projection
apparatus in FIG. 8.
[0031] {FIG. 10} FIG. 10 is an overall configuration diagram
schematically showing a hologram-image projection apparatus
according to a fourth embodiment of the present invention.
[0032] {FIG. 11} FIG. 11 is a flowchart for explaining a
hologram-image projection method according to the fourth embodiment
of the present invention, using the hologram-image projection
apparatus in FIG. 10.
[0033] {FIG. 12A} FIG. 12A is a diagram showing a focal point map
when it is assumed that the objective lens is aberration-free, in
the hologram-image projection method in FIG. 11.
[0034] {FIG. 12B} FIG. 12B is a diagram showing a virtual image at
the focal plane, in the hologram-image projection method in FIG.
11.
[0035] {FIG. 13} FIG. 13 is an overall configuration diagram
schematically showing a hologram-image projection apparatus
according to a fifth embodiment of the present invention.
[0036] {FIG. 14} FIG. 14 is an overall configuration diagram
showing a modification of the hologram-image projection apparatus
in FIG. 13.
DESCRIPTION OF EMBODIMENTS
[0037] A hologram-image projection apparatus and a hologram-image
projection method according to a first embodiment of the present
invention will be described below with reference to FIG. 1 and FIG.
2.
[0038] As shown in FIG. 1, a hologram-image projection apparatus 1
according to this embodiment, which is a microscope system,
includes a light source apparatus 2 that emits laser light, a
microscope apparatus 3 that irradiates a specimen A with laser
light incident from the light source apparatus 2, and a control
apparatus 4 that controls the wavefront of the laser light entering
the microscope apparatus 3 from the light source apparatus 2.
[0039] The light source apparatus 2 includes a laser light source 5
that generates laser light, a collimator lens 6 that converts the
laser light emitted from the laser light source 5 into a collimated
beam, a wavefront modulating section 7 that modulates the wavefront
of the collimated beam of laser light, relay lenses 8 and 10, and a
scanner 9 that scans the laser light.
[0040] The wavefront modulating section 7 includes a prism 11 that
reflects the laser light and a reflective wavefront modulating
device 12 that reflects the laser light reflected by the prism 11,
during which time the wavefront of the laser light is modulated by
a phase pattern, and returns the laser light to the prism 11.
[0041] The laser light reflected by the prism 11 has its light path
folded by the wavefront modulating device 12 so as to return to the
same prism 11 and then returns to a light path along the same axis
as the laser light from the laser light source 5.
[0042] The wavefront modulating device 12 is formed of a segmented
MEMS mirror whose surface shape can be arbitrarily changed by the
control apparatus 4, to be described later. In this case, the
surface shape, which is formed of the depressions and protrusions
of the individual segments in the MEMS mirror, forms a phase
pattern for modulating the wavefront of the laser light. The
wavefront modulating device 12 and an entrance pupil position of an
objective lens 13 are disposed in an optically conjugate positional
relationship.
[0043] The scanner 9 is a so-called proximity galvanometer mirror
in which two galvanometer mirrors 9a and 9b that can be swiveled
about axes disposed in mutually orthogonal directions are disposed
in proximity to each other and can two-dimensionally scan the
incident laser light.
[0044] The microscope apparatus 3 includes the objective lens 13,
which focuses the laser light onto the specimen A, disposed on a
stage 14, and which also collects light coming from the specimen A;
a light detector 15, formed of a photomultiplier tube, for
detecting the light collected by the objective lens 13; a camera
16, such as a CCD, that captures a fluorescence image in the
specimen A; and a mirror 17 that is inserted in and removed from
the light path so as to switch the light path to either the light
detector 15 or the camera 16. Reference numerals 18 to 20 are
focusing lenses, and reference numeral 21 is a dichroic mirror. The
objective lens 13 has known characteristic data related to the
overall system and is provided in such a manner that the distance
between the objective lens 13 and the stage 14 in the optical axis
direction can be varied. The overall characteristic data of the
objective lens 13 includes the focal length, the numerical
aperture, the entrance pupil diameter, etc. of the overall
objective lens 13.
[0045] By setting the surface shape of the wavefront modulating
device 12 to a flat reflective surface shape, it is possible to
make laser light having a plane-wave wavefront incident at the
entrance pupil position of the objective lens 13. Accordingly, it
is possible to focus the laser light at the focal plane of the
objective lens 13.
[0046] With the mirror 17 switched to the light detector 15 side (a
position where it is removed from the light path, as indicated in
the dotted lines), the laser light is emitted from the laser light
source 5, and while the laser light focused at the focal plane in
the specimen A is two-dimensionally scanned by driving the scanner
9, fluorescence produced at each focal position is detected by the
light detector 15, thereby enabling acquisition of a
two-dimensional fluorescence image of the specimen A extending over
the focal plane of the objective lens 13.
[0047] Then, by acquiring a plurality of two-dimensional
fluorescence images (slice images) while changing the position of
the focal plane of the objective lens 13 by changing the relative
distance between the objective lens 13 and the stage 14, it is
possible to acquire a three-dimensional fluorescence image of the
specimen A.
[0048] As shown in FIG. 1, the control apparatus 4 includes a
storage section 22 that stores the overall characteristic data of
the objective lens 13 and the refractive index of the specimen A;
an input section (position-information setting section) 23 for
setting three-dimensional position information of focal points of
the laser light in the specimen A; a wavefront calculating section
24 that calculates the wavefront at the entrance pupil position of
the objective lens 13, for each focal point, on the basis of the
position information of each focal point set via the input section
23, as well as the overall characteristic data of the objective
lens 13 and the refractive index of the specimen A stored in the
storage section 22; a wavefront combining section 25 that combines
the wavefronts calculated for all focal points; and a phase-pattern
setting section 26 that sets a phase pattern to be applied to the
wavefront modulating device 12 on the basis of the combined
wavefront combined by the wavefront combining section 25.
[0049] The input section 23 is configured to set three-dimensional
position information for each focal position by the user specifying
a position where the laser light is desired to be focused, that is,
the focal point, on a monitor (not illustrated). The
depthwise-direction position information of each focal point is set
in the form of relative depth information from a reference plane,
when a plane at a prescribed depth in the specimen is assumed as
the reference surface. This reference plane is preferably set to
the focal plane of the objective lens 13 when the objective lens 13
is fixed at a prescribed position relative to the specimen A;
however, it is not limited thereto.
[0050] The focal point in the specimen A may be set to an arbitrary
position, or it may be set to a position that responds to a light
stimulus. When it is desired to set the focal point to a position
responding to a light stimulus, in order that the observer can
easily specify the focal point, it is preferable to display a
two-dimensional or three-dimensional image acquired by the
microscope apparatus 3 on the monitor (not shown).
[0051] For each focal point that is set, the wavefront calculating
section 24 calculates the wavefront corresponding to each focal
point by using the position information set via the input section
23, as well as the overall characteristic data of the objective
lens 13 and the refractive index of the specimen A stored in the
storage section 22.
[0052] Specifically, a point light source is assumed at a position
specified by the position information set for the focal point, and
the wavefront is calculated by performing reverse ray tracing of
the laser light from that point light source to the entrance pupil
position of the objective lens 13, using the refractive index of
the specimen A and the overall characteristic data of the objective
lens 13. For focal points disposed on the focal plane serving as
the reference plane, the wavefronts at the entrance pupil position
are plane waves whose angles relative to the optical axis of the
objective lens 13 differ according to the positions of the focal
points in the reference plane.
[0053] The wavefront combining section 25 is configured to combine
the wavefronts calculated for all focal points. In this embodiment,
the wavefront combining section 25 is configured to calculate the
linear summation of the wavefronts calculated for all focal
points.
[0054] The phase-pattern setting section 26 sets a phase pattern to
be applied to the wavefront modulating device 12 on the basis of
the obtained combined wavefront at the entrance pupil position of
the objective lens 13 and outputs the phase pattern to the
wavefront modulating device 12. Because the wavefront modulating
device 12 is in a conjugate relationship with the entrance pupil of
the objective lens 13, the phase pattern applied to the wavefront
modulating device 12 is identical to the combined wavefront at the
entrance pupil position of the objective lens 13 or is a pattern
formed by phase-wrapping processing. In phase-wrapping processing,
when the range of phase modulation at the wavefront modulating
device 12 is set to 2 n.pi. (n is an integer), for portions in the
combined wavefront at the entrance pupil position of the objective
lens 13 that have a phase difference exceeding 2 n.pi., 2 n.pi. is
subtracted from that phase difference. With the segmented MEMS
mirror in this embodiment, because the range of phase modulation is
normally set to a range of 2 n.pi., phase-wrapping processing is
performed as required.
[0055] In this way, the wavefront modulating device 12 is adjusted
so as to assume a surface shape matching the input phase pattern.
Thus, the laser light reflected at the wavefront modulating device
12 is modulated at the surface of the wavefront modulating device
12 to have the same wavefront as the obtained combined wavefront at
the entrance pupil position of the objective lens 13. Therefore, by
focusing such laser light with the objective lens 13, it is
simultaneously focused at the individual set focal points while
keeping the objective lens 13 fixed.
[0056] A hologram-image projection method for projecting a hologram
image like one in which laser light is focused simultaneously at a
plurality of positions at different depths in the specimen A by
using the thus-configured hologram-image projection apparatus 1
according to this embodiment will now be described.
[0057] First, as shown in FIG. 2, position information for each
focal point is set via the input section 23 (step S1).
[0058] Here, when the focal point is set to a position that
responds to a light stimulus in the specimen A, the wavefront
modulating device 12 is set to a phase pattern forming a flat
reflective surface shape based on the output from the control
apparatus 4. In the microscope apparatus 3, the mirror 17 is
retracted from the light path. Then, laser light is emitted from
the laser light source 5, and the laser light is two-dimensionally
scanned by the scanner 9.
[0059] The laser light emitted from the laser light source 5
propagates along the light path without changing its wavefront and,
after being two-dimensionally scanned by the scanner 9, is
reflected by the dichroic mirror 21 to enter the objective lens 13
and is focused at the focal plane in the specimen A. Fluorescence
is produced at the focal position of the laser light, and the
fluorescence produced is collected by the objective lens 13, is
transmitted through the dichroic mirror 21, is focused by the
focusing lenses 18 and 19, and is detected by the light detector
15.
[0060] Because the fluorescence is produced only in an extremely
shallow region in the vicinity of the focal plane of the objective
lens 13, by storing the intensity of the fluorescence detected by
the light detector 15 and the scanning position of the laser light
scanned by the scanner 9 in association with each other, it is
possible to obtain a fluorescence image (slice image) of the
specimen A extending over the focal plane. By obtaining a plurality
of slice images while moving the objective lens 13 and the specimen
A relative to each other in the optical axis direction, it is
possible to obtain a three-dimensional fluorescence image.
[0061] In the three-dimensional fluorescence image displayed on the
monitor (not shown), the observer specifies the positions of the
focal points where the laser light should be focused. For example,
if the specimen A is a nerve cell whose behavior is to be observed
when stimulated with laser light, the focal points where the
stimulus is to be applied are distributed three-dimensionally. In
this case, the observer sets the three-dimensional position
information of the focal points by specifying all focal points.
[0062] By fixing the objective lens 13 relative to the specimen A,
the focal plane of the objective lens 13 is fixed relative to the
specimen. Regarding the depth information in the position
information of the focal points, the relative distance in the
depthwise direction from the reference plane, when a plane at a
prescribed depth in the specimen A is defined as the reference
plane, is set. This reference plane is set, for example, to the
focal plane of the objective lens 13.
[0063] On the other hand, when the focal plane is set to an
arbitrary position in the specimen A, it is not necessary to
acquire such a three-dimensional fluorescence image.
[0064] Next, reverse ray tracing is performed in the wavefront
calculating section 24 using the position information set via the
input section 23, as well as the overall characteristic data of the
objective lens 13 and the refractive index of the specimen A stored
in the storage section 22, and the wavefront at the entrance pupil
position of the objective lens 13 is calculated for the laser light
emitted from the point light source assumed at each focal position
(step S2).
[0065] Once the wavefronts at the entrance pupil position of the
objective lens 13 are calculated for all focal points, a linear
summation of the wavefronts is calculated by the wavefront
combining section 25, thus generating a combined wavefront (step
S3). The combined wavefront thus generated is input to the
phase-pattern setting section 26, and the phase pattern to be
applied to the wavefront modulating device 12 is set to be
identical to the combined wavefront at the entrance pupil position
of the objective lens 13 or a pattern formed by phase-wrapping
processing (step S4). Then, the phase pattern set by the
phase-pattern setting section 26 is output to the wavefront
modulating device 12.
[0066] This completes the preparations for performing observation
while applying a light stimulus to the specimen A.
[0067] In this state, the microscope apparatus 3 is configured so
that the mirror 17 is inserted into the light path (disposed at a
position indicated by the solid lines in the figure), and the
fluorescence collected by the objective lens 13 is focused by the
focusing lens 20 and is acquired by the camera 16. Then, with the
scanner 9 stopped at the origin, when the laser light emitted from
the laser light source 5 is introduced into the wavefront
modulating section 7, the wavefront of the laser light is modulated
according to the phase pattern displayed on the wavefront
modulating device 12. The modulated laser light passes through the
relay lenses 8, the scanner 9, and the relay lenses 10, is
reflected by the dichroic mirror 21, and enters the entrance pupil
of the objective lens 13 (step S5).
[0068] Because the entrance pupil position of the objective lens 13
is located at a position that is optically conjugate with the
wavefront modulating device 12, the laser light incident at the
entrance pupil position has a wavefront identical to the wavefront
at the instant it is modulated by the wavefront modulating device
12.
[0069] Then, by focusing this laser light with the objective lens
13, a three-dimensional hologram image like one in which laser
light is focused simultaneously at a plurality of focal points
located at different positions in the depthwise direction can be
projected inside the specimen A.
[0070] By simultaneously irradiating a plurality of specified focal
points with the laser light, in the state where a stimulus is
applied to the specimen A, the fluorescence emitted from the
specimen A is collected by the objective lens 13, is transmitted
through the dichroic mirror 21, is reflected by the mirror 17, is
focused by the focusing lens 20, and is acquired by the camera 16.
Thus, it is possible to obtain a fluorescence image of the specimen
A while a light stimulus is applied thereto.
[0071] Thus, with the hologram-image projection apparatus 1 and the
hologram-image projection method according to this embodiment, it
is possible to focus laser light simultaneously at a plurality of
sites of interest located at different positions in the depthwise
direction of the specimen A. Consequently, when the focal points
are set at positions responding to a light stimulus in the specimen
A, an advantage is afforded in that it is possible to correctly
observe the behavior of the specimen occurring directly after the
light stimulus without any time delay.
[0072] Note that, in this embodiment, although wavefront combining
is performed in the wavefront combining section 25 by calculating a
linear summation of the wavefronts, instead of this, it may be
performed by arraying divided regions of a plurality of wavefronts
calculated for the individual focal points with a ratio equal to
the reciprocal of the total number of focal points.
[0073] More specifically, if n is the total number of focal points,
a linear summation of the wavefronts calculated for the individual
focal points is not calculated, but the region on the wavefront
modulating device 12 is divided, and any of the plurality of focal
points are associated with the individual divided regions. The
associating process here is preferably performed so that the
regions associated with the individual focal points are
periodically distributed with a ratio equal to the reciprocal of
the total number of focal points, n. Also, the distribution of the
regions associated with the individual focal points may be in the
form of a mosaic or may be in form of concentric circles. Assuming
this kind of association, in the wavefronts of the individual focal
points which are calculated in advance, wavefront elements of
portions of the wavefront modulating device 12 overlapping the
associated regions are extracted, and the wavefronts are combined
by arraying the extracted wavefront elements over the entire
region. The phase pattern to be applied to the wavefront modulating
device 12 is set on the basis of the combined wavefront obtained in
this way.
[0074] With the hologram-image projection method of this
embodiment, the three-dimensional position information of each of
the focal points three-dimensionally located at different positions
in the specimen A is set in the position setting step S1, and in
the wavefront calculating step S2, reverse ray tracing is performed
from each focal point to the entrance pupil position of the
objective lens 13. By combining the plurality of wavefronts
calculated in the wavefront calculating step S2 in the wavefront
combining step S3, a combined wavefront serving as the wavefront
required to be incident at the entrance pupil position of the
objective lens 13 to simultaneously focus the laser light at
different three-dimensional positions is obtained by calculation.
Then, a phase pattern is applied to the wavefront modulating device
12 so that the wavefront of the laser light incident on the
wavefront modulating device 12 is modulated to a wavefront
identical to the obtained combined wavefront. Accordingly, a
hologram image like one in which laser light is focused
simultaneously at focal points disposed three-dimensionally at
different depth positions can be projected inside the specimen
A.
[0075] With the hologram-image projection apparatus 1 of this
embodiment, once the three-dimensional position information of the
individual focal points is set via the input section 23, reverse
ray tracing from the focal points to the entrance pupil position of
the objective lens 13 is performed by the wavefront calculating
section 24 using the set position information, as well as the
refractive index of the specimen A and the overall characteristic
data of the objective lens 13. Accordingly, the wavefront at the
entrance pupil position of the objective lens 13 is calculated for
the laser light coming from each focal point. Then, the plurality
of wavefronts obtained for the individual focal points are combined
in the wavefront combining section 25 to obtain the combined
wavefront. The phase pattern is set in the phase-pattern setting
section 26 on the basis of the combined wavefront obtained in this
way. Accordingly, merely by radiating the laser light on the
wavefront modulating device 12 to which the set phase pattern has
been applied, a hologram image that causes the laser light focused
by the objective lens 13 to be simultaneously focused at a
plurality of focal points disposed at different positions
three-dimensionally can be projected inside the specimen.
[0076] A hologram-image projection apparatus and a hologram-image
projection method according to a second embodiment of the present
invention will be described below with reference to FIGS. 3 to 6
and FIGS. 7A to 7C.
[0077] In this embodiment, elements having the same configuration
as those in the hologram-image projection apparatus and the
hologram-image projection method according to the above-described
first embodiment are assigned the same reference numerals, and a
description thereof is omitted.
[0078] In the first embodiment, the wavefront is generated by
performing reverse ray tracing from each of the set focal points to
the entrance pupil position of the objective lens 13. In this
embodiment, however, instead of this, a spot image is obtained by
performing forward ray tracing from each focal point to a
prescribed reference plane, and the wavefront is generated by
transforming this image.
[0079] As shown in FIG. 3, a hologram-image projection apparatus
100 according to this embodiment differs from the first embodiment
in the configuration of the control apparatus 4. Specifically, the
control apparatus 4 includes a storage section 22 that stores the
overall characteristic data of an objective lens 13 and the
refractive index of a specimen A; an input section
(position-information setting section) 23 for inputting
three-dimensional position information of the focal point of the
laser light in the specimen A; a virtual-image creating section 27
that creates a virtual image formed by projecting each focal point
on a prescribed reference plane on the basis of the overall
characteristic data of the objective lens 13 and the refractive
index of the specimen A stored in the storage section 22; a
wavefront calculating section 28 that calculates the wavefront at
the entrance pupil position of the objective lens 13 by Fourier
transforming the created virtual image; and a phase-pattern setting
section 26 that sets the phase pattern to be applied to a wavefront
modulating device 12 on the basis of the wavefront calculated by
the wavefront calculating section 28.
[0080] Here, the wavefront calculating section 28 Fourier
transforms the virtual image created by the virtual-image creating
section 27 using the overall characteristic data of the objective
lens 13 and the refractive index of the specimen A stored in the
storage section 22.
[0081] A hologram-image projection method for projecting a hologram
image like one in which laser light is focused simultaneously at a
plurality of positions at different depths in the specimen A by
using the thus-configured hologram-image projection apparatus 100
according to this embodiment will be described.
[0082] First, as shown in FIG. 4, plane position information of
each focal point in the reference plane is set at the input section
23 (step S11).
[0083] Next, on the basis of the plane position information of each
focal point in the reference plane set via the input section 23, a
first virtual image in which it is assumed that all focal points
disposed at different positions in the depthwise direction relative
to the reference plane form a spot by focusing light on the
reference plane is created at the virtual-image creating section 27
(step S12). The first virtual image is created by specifying each
focal point using only a single slice image acquired at an
arbitrary reference plane. Although this reference plane is
preferably set at the focal plane of the objective lens 13, it is
not limited thereto. Thus, because no depth information of the
actual focal points is used in creating the first virtual image,
the first virtual image does not reflect a real spot image, but is
a spot image in which it is assumed that all focal points are at
the reference plane.
[0084] Next, for each of the focal points, depth information from
the reference plane is input via the input section 23 (step
S13).
[0085] Then, a second virtual image at the reference plane is
created by performing correction of spots corresponding to the
focal points focused at different positions from the reference
plane to add the depth information set via the input section 23
(step S14). After this, the created second virtual image is Fourier
transformed, and a wavefront, at the entrance pupil position of the
objective lens 13, that enables projection of the spots in the
second virtual image, is calculated (step S15). Then, the
calculated wavefront is input to the phase-pattern setting section
26, and the phase pattern applied to the wavefront modulating
device 12 is set to be identical to the wavefront at the entrance
pupil position of the objective lens 13 or to a pattern obtained by
phase-wrapping processing (step S16). Then, the phase pattern set
by the phase-pattern setting section 26 is output to the wavefront
modulating device 12. In this state, when laser light is emitted
from the laser light source 5 and enters the wavefront modulating
section 7, the wavefront of the laser light is modulated according
to the phase pattern on the wavefront modulating device 12. The
modulated laser light passes through the relay lenses 8, the
scanner 9, the relay lenses 10, and the dichroic mirror 21 and is
focused onto the specimen by the objective lens 13 to irradiate it
(step S17).
[0086] In the method of creating the second virtual image, a point
light source is assumed at a focal point P that is not located on
the reference plane Q, and by performing forward ray tracing from
that point source to the reference plane Q, as shown in FIG. 5A, or
by performing reverse ray tracing, as shown in FIG. 5B, a wavefront
that forms a new spot R1 or R2 on the reference plane Q is
calculated. For the obtained wavefront, at the spot R1 in the case
of forward ray tracing, shown in FIG. 6A, and the spot R2 in the
case of reverse ray tracing, shown in FIG. 6B, depth information
contained in the wavefront is inverted. Correction is applied to
the spots R on the reference plane Q.
[0087] In another method of creating the second virtual image, as
shown in FIG. 7A, the objective lens 13 is fixed relative to the
specimen A, the laser light is focused on the reference plane Q,
and a fluorescence image is acquired. Next, as shown in FIG. 7B,
the objective lens 13 is moved by distance Ad in the optical axis
direction. Accordingly, because the focal point P is shifted from
the reference plane Q, the intensity of the fluorescence image
varies. In this state, as shown in FIG. 7C, the intensity of the
fluorescence image is observed when the coefficient Z.sub.4 in a
fourth-order Zernike polynomial, which is vector information
representing the aberrations at the focal plane Q of the objective
lens 13, is continuously varied, and the value of the coefficient
Z.sub.4 that yields the same intensity as the initial intensity is
determined. When the change in the coefficient Z.sub.4 at this time
is .DELTA.Z, the proportionality coefficient K=.DELTA.Z/.DELTA.d is
calculated. Accordingly, the Zernike coefficient Z.sub.4 and a
wavefront W at the entrance pupil position (the position indicated
by the black triangles in FIGS. 7A, 7B, and 7C) of the objective
lens 13 are calculated on the basis of the calculated
proportionality coefficient K and the depth information from the
reference plane Q to the focal point P.
[0088] A cylindrical function w(r, .theta.) giving the shape of the
wavefront, which is a Zernike polynomial, is shown in the following
formula:
w ( r , .theta. ) = Z 1 + Z 2 .rho.cos .theta. + Z 3
.rho.sin.theta. + Z 4 ( 2 .rho. 2 - 1 ) + Z 5 .rho. 2 cos 2 .theta.
+ Z 6 .rho. 2 sin 2 .theta. + Z 7 ( 3 .rho. 2 - 2 ) .rho. cos
.theta. + Z 8 ( 3 .rho. 2 - 2 ) .rho.sin .theta. ##EQU00001##
Here, .rho. is the distance from the center of the pupil normalized
by the radius from the center of the entrance pupil of the
objective lens 13, and .theta. is the angle relative to a
prescribed reference line in polar coordinates in the plane of the
wavefront modulating device 12 or the entrance pupil plane of the
objective lens 13. Zn are Zernike coefficients.
[0089] By using the fourth term, which is a defocus term, in the
Zernike polynomial, the wavefront W at the entrance pupil position
of the objective lens 13 can be expressed by
W=Z.sub.4.times.(2.rho..sup.2-1).
The Zernike coefficient Z.sub.4 can be expressed by
Z.sub.4=K.times.d.
[0090] Here, d is the depth information from the reference plane Q
to the focal point P, set via the input section 23.
[0091] After the wavefront W is obtained in this way, the wavefront
at the reference plane Q when laser light with this wavefront is
made incident at the entrance pupil position of the objective lens
13 is determined and is corrected by substituting the corresponding
spot in the first virtual image with this wavefront. The second
virtual image is created by repeating the same operation for all
focal points P shifted in the depthwise direction relative to the
reference plane Q.
[0092] In this embodiment, after creating the first virtual image
for when it is assumed that all focal points are focused on the
reference plane, the second virtual image corrected on the basis of
the depth information is created. Instead of this, however, a
virtual image in which the individual focal points are projected on
a prescribed reference image on the basis of three-dimensional
position information of the focal points may be created in one
step. In this case, the three-dimensional position information of
the individual focal points is set via the input section 23. Also,
the virtual-image creating section 27 creates a virtual image in
which the individual focal points are projected on a prescribed
reference plane on the basis of this three-dimensional position
information, as well as the overall characteristic data of the
objective lens 13 and the refractive index of the specimen stored
in the storage section 22.
[0093] With the hologram-image projection method of this
embodiment, the three-dimensional position information of the
individual focal points three-dimensionally disposed at different
positions in the specimen A is set in the position setting steps
S11 and S13, and a virtual image in which a plurality of focal
points to be formed by focusing the laser light via the objective
lens 13 are projected on a prescribed reference plane is created in
the virtual-image creating steps S12 and S14. In the wavefront
calculating step S15, the wavefront at the entrance pupil position
of the objective lens 13 is calculated by Fourier transforming the
virtual image. Then, the phase pattern to be applied to the
wavefront modulating device 12 is set in the phase-pattern setting
step S16, and in a focusing step, the set phase pattern is applied
to the wavefront modulating device 12, and the laser light is made
incident on the wavefront modulating device 12, thereby focusing
the laser light, whose wavefront has been modulated by the phase
pattern, on the specimen A via the objective lens 13. Accordingly,
a hologram image in which the laser light is focused simultaneously
at a plurality of focal points disposed three-dimensionally in the
specimen A can be projected in the specimen A.
[0094] In addition, of the spots in the first virtual image, in
which all spots are focused at the prescribed reference plane, for
spots corresponding to focal points that are not focused at the
reference plane, because the phase pattern is generated from the
second virtual image created by correcting the spots on the basis
of the set depth information, merely by radiating a collimated beam
of laser light onto the wavefront modulating device 12 to which
this phase pattern is applied, the laser light modulated by the
wavefront modulating device 12 can be focused even at depth
positions in the specimen that differ from the reference plane.
Accordingly, it is possible to focus the laser light simultaneously
at a plurality of different focal points in the depthwise
direction.
[0095] By replacing the spots in the first virtual image with spots
obtained by forward ray tracing or reverse ray tracing of the laser
light focused at each focal point up to the reference plane, it is
possible to more easily calculate the second virtual image that
forms spots such that they are focused at different positions in
the depthwise direction inside the specimen.
[0096] In addition, at the position of the objective lens 13 for
focusing the laser light at the reference plane, it is possible to
obtain vector information representing the aberrations at the focal
plane of the objective lens 13 necessary for focusing the laser
light at depth positions that differ from the reference plane by
multiplying the depth information representing the actual distance
between the focal points P and the reference plane Q by the
calculated proportionality coefficient K. By creating the second
virtual image in which the spots are corrected on the basis of this
vector information, it is possible to focus the laser light
simultaneously at a plurality of different focal points in the
depthwise direction while keeping the objective lens 13 fixed.
[0097] With the hologram-image projection apparatus 100 of this
embodiment, when the three-dimensional position information of the
individual focal points is set via the input section 23, a virtual
image in which the individual focal points are projected on the
prescribed reference plane inside the specimen A is created by the
virtual-image creating section 27 by using this set position
information, and the wavefront at the entrance pupil position of
the objective lens 13 is calculated by the wavefront calculating
section by Fourier transforming the virtual image using the
refractive index of the specimen A and the overall characteristic
data of the objective lens 13. Then, the phase pattern to be
applied to the wavefront modulating device 12 is set on the basis
of the calculated wavefront. Accordingly, simply by radiating laser
light onto the wavefront modulating device 12 to which the set
phase pattern has been applied, a hologram image in which the laser
light focused by the objective lens 13 is simultaneously focused at
a plurality of focal points disposed at different three-dimensional
positions can be projected inside the specimen.
[0098] In the first and second embodiments described above,
although a segmented MEMS mirror whose surface shape can be varied
has been illustrated as an example of the wavefront modulating
device 12, instead of this, any other type of wavefront modulating
device 12 can be used, for example, a liquid crystal device, a
deformable mirror, or the like. In the case of a liquid crystal
device, the refractive index distribution due to the alignment of
the liquid crystal molecules constitutes the phase pattern, and in
the case of a deformable mirror, the surface shape thereof
constitutes the phase pattern.
[0099] A hologram-image projection apparatus and a hologram-image
projection method according to a third embodiment of the present
invention will be described below with reference to FIGS. 8 and
9.
[0100] As show in FIG. 8, a hologram-image projection apparatus 101
according to this embodiment, which is a microscope system,
includes a light source apparatus 102 that emits laser light, a
microscope apparatus 103 that irradiates a specimen A with the
laser light incident from the light source apparatus 102, and a
control apparatus 104 that adjusts the laser light entering the
microscope apparatus 103 from the light source apparatus 102.
[0101] The light source apparatus 102 includes a laser light source
105 that emits laser light, a collimator lens 106 that converts the
laser light emitted from the laser light source 105 into a
collimated beam, a wavefront modulating section 107 that modulates
the wavefront of the collimated beam of laser light, relay lenses
108 and 110, and a scanner 109 that scans the laser light.
[0102] The wavefront modulating section 107 includes a prism 111
that reflects the laser light and a reflective wavefront modulating
device 112 that reflects the laser light reflected by the prism
111, during which time the wavefront of the laser light is
modulated by a phase pattern, and returns the modulated laser light
to the prism 111.
[0103] The laser light reflected by the prism 111 has its light
path folded at the wavefront modulating device 112 so as to return
to the same prism 111, and then returns to a light path on the same
axis as the laser light from the laser light source 105.
[0104] The wavefront modulating device 112 is formed of a segmented
MEMS mirror whose surface shape can be arbitrarily changed by the
control apparatus 104, described later. In this case, the surface
shape formed by the indentations and protrusions of the individual
segments of the MEMS mirror constitutes the phase pattern for
modulating the wavefront of the laser light. The wavefront
modulating device 112 and the entrance pupil position of the
objective lens 113 are disposed in an optically conjugate
positional relationship.
[0105] The scanner 109 is a so-called proximity galvanometer mirror
in which two galvanometer mirrors 109a and 109b that can be
swiveled about axes disposed in mutually intersecting directions
are placed in close proximity to each other, so that the incident
laser light can be scanned two-dimensionally.
[0106] The microscope apparatus 103 includes the objective lens
113, which focuses the laser light onto the specimen A, disposed on
a stage 114, and also collects light coming from the specimen A; a
light detector 115, formed of a photomultiplier tube, for detecting
fluorescence collected by the objective lens 113; a camera 116,
such as a CCD, that captures a fluorescence image in the specimen
A; and a mirror 117 that is inserted in and removed from the light
path so as to switch the light path to either the light detector
115 or the camera 116. Reference numerals 118 to 120 are focusing
lenses, and reference numeral 121 is a dichroic mirror. The
objective lens 113 has known lens data for the individual lenses
constituting the objective optical system and is provided in such a
manner that the distance between the objective lens 113 and the
stage 114 in the optical axis direction can be varied.
[0107] By setting the surface shape of the wavefront modulating
device 112 to a flat reflective surface shape, it is possible to
make laser light having a plane-wave wavefront incident at the
entrance pupil position of the objective lens 113. Accordingly, it
is possible to focus the laser light at the focal plane of the
objective lens 113.
[0108] With the mirror 117 switched to the light detector 115 side
(a position where it is removed from the light path, as indicated
in the dotted lines), the laser light is emitted from the laser
light source 105, and while the laser light focused at the focal
plane in the specimen A is two-dimensionally scanned by driving the
scanner 109, fluorescence produced at each focal position is
detected by the light detector 115, thereby enabling acquisition of
a two-dimensional fluorescence image of the specimen A extending
over the focal plane of the objective lens 113.
[0109] As shown in FIG. 8, the control apparatus 104 includes a
storage section 122 that stores the lens data of the objective lens
113; an input section (position-information setting section) 123
for setting position information of focal points of the laser light
in the specimen A; a wavefront calculating section 124 that
calculates the wavefront at the entrance pupil position of the
objective lens 113 corresponding to each focal point on the basis
of the position information of each focal point set via the input
section 123, as well as the lens data of each lens constituting the
objective lens 113 and the refractive index of the specimen A
stored in the storage section 122; a wavefront combining section
125 that combines a plurality of wavefronts calculated for all
focal points; and a phase-pattern setting section 126 that sets a
phase pattern to be applied to the wavefront modulating device 112
on the basis of the combined wavefront combined by the wavefront
combining section 125.
[0110] The input section 123 is configured to set two-dimensional
position information for each focal point by the user specifying a
position where the laser light is desired to be focused, that is,
the focal point, on a monitor (not illustrated). The focal point in
the specimen A may be set to an arbitrary position, or it may be
set to a position that responds to a light stimulus. When it is
desired to set the focal point to a position that responds to a
light stimulus, in order that the user can easily specify the focal
point, it is preferable to display a two-dimensional image acquired
by the microscope apparatus 103 on the monitor (not shown).
[0111] For each focal point that is set, the wavefront calculating
section 124 calculates the wavefront corresponding to each focal
point by using the position information set via the input section
123 and the lens data of the objective lens 113 stored in the
storage section 122.
[0112] Specifically, a point light source is assumed at a position
specified by the position information set for the focal point, and
the wavefront is calculated by performing reverse ray tracing of
the laser light from that point light source to the entrance pupil
position of the objective lens 113, using the lens data of the
objective lens 113.
[0113] The wavefront combining section 125 is configured to combine
the wavefronts calculated for all focal points. In this embodiment,
the wavefront combining section 125 is configured to calculate the
linear summation of the wavefronts calculated for all focal
points.
[0114] The phase-pattern setting section 126 sets a phase pattern
to be applied to the wavefront modulating device 112 on the basis
of the combined wavefront obtained at the entrance pupil position
of the objective lens 113 and outputs the phase pattern to the
wavefront modulating device 112. Because the wavefront modulating
device 112 is in a conjugate relationship with the entrance pupil
of the objective lens 113, the phase pattern applied to the
wavefront modulating device 112 is identical to the combined
wavefront at the entrance pupil position of the objective lens 113
or is a pattern formed by phase-wrapping processing. In
phase-wrapping processing, when the range of phase modulation at
the wavefront modulating device 112 is set to 2 n.pi. (n is an
integer), for portions in the combined wavefront at the entrance
pupil position of the objective lens 113 that have a phase
difference exceeding 2 n.pi., 2 n.pi. is subtracted from that phase
difference. With the segmented MEMS mirror in this embodiment,
because the range of phase modulation is normally set to a range of
2 n.pi., phase-wrapping processing is performed as required.
[0115] In this way, the wavefront modulating device 112 is adjusted
so as to assume a surface shape matching the input phase pattern.
Thus, the laser light reflected at the wavefront modulating device
112 is modulated at the surface of the wavefront modulating device
112 to have the same wavefront as the combined wavefront obtained
at the entrance pupil position of the objective lens 113.
Therefore, by focusing this laser light with the objective lens
113, it is simultaneously focused at the individual set focal
points while keeping the objective lens 113 fixed.
[0116] A hologram-image projection method for projecting a hologram
image like one in which laser light is focused simultaneously at a
plurality of different positions in the specimen A by using the
thus-configured hologram-image projection apparatus 101 according
to this embodiment will now be described.
[0117] First, as shown in FIG. 9, position information for each
focal point is set via the input section 123 (step S101).
[0118] Here, when the focal point is set to a position that
responds to a light stimulus in the specimen A, the wavefront
modulating device 112 is set to a phase pattern forming a flat
reflective surface shape based on the output from the control
apparatus 104. In the microscope apparatus 103, the mirror 117 is
retracted from the light path. Then, laser light is emitted from
the laser light source 105, and the laser light is
two-dimensionally scanned by the scanner 109.
[0119] The laser light emitted from the laser light source 105
propagates along the light path without changing its wavefront and,
after being two-dimensionally scanned by the scanner 109, is
reflected by the dichroic mirror 121 to enter the objective lens
113 and is focused at the focal plane in the specimen A.
Fluorescence is produced at the focal position of the laser light,
and the fluorescence produced is collected by the objective lens
113, is transmitted through the dichroic mirror 121, is focused by
the focusing lenses 118 and 119, and is detected by the light
detector 115.
[0120] Because the fluorescence is produced only in an extremely
shallow region in the vicinity of the focal plane of the objective
lens 113, by storing the intensity of the fluorescence detected by
the light detector 115 and the scanning position of the laser light
scanned by the scanner 109 in association with each other, it is
possible to obtain a fluorescence image (slice image) of the
specimen A extending over the focal plane.
[0121] In the two-dimensional fluorescence image displayed on the
monitor (not shown), the observer specifies the positions of the
focal points where the laser light should be focused. For example,
if the specimen A is a nerve cell whose behavior is to be observed
when stimulated with laser light, the focal points where the
stimulus is to be applied are distributed. In this case, the
observer sets the two-dimensional positions of the focal points by
specifying all focal points.
[0122] On the other hand, when the focal points are set to
arbitrary positions in the specimen A, it is not necessary to
acquire such a two-dimensional fluorescence image.
[0123] Reverse ray tracing is performed in the wavefront
calculating section 124 using the position information set via the
input section 123, as well as the lens data of each lens
constituting the objective lens 113 and the refractive index of the
specimen A stored in the storage section 122, and the wavefront at
the entrance pupil position of the objective lens 113 is calculated
for the laser light emitted from a point light source assumed at
each focal point (step S102).
[0124] Once the wavefronts at the entrance pupil position of the
objective lens 113 are calculated for all focal points, a linear
summation of the wavefronts is calculated by the wavefront
combining section 125, thus forming a combined wavefront (step
S103). The combined wavefront thus formed is input to the
phase-pattern setting section 126, and the phase pattern to be
applied to the wavefront modulating device 112 is set to be
identical to the combined wavefront at the entrance pupil position
of the objective lens 113 or a pattern formed by phase-wrapping
processing (step S104). Then, the phase pattern set by the
phase-pattern setting section 126 is output to the wavefront
modulating device 112.
[0125] This completes the preparations for performing observation
while applying a light stimulus to the specimen A.
[0126] In this state, the microscope apparatus 103 is configured so
that the mirror 117 is inserted into the light path (disposed at a
position indicated by the solid lines in the figure), and the
fluorescence collected by the objective lens 113 is focused by the
focusing lens 120 and is acquired by the camera 116. Then, with the
scanner 109 stopped at the origin, when the laser light emitted
from the laser light source 105 is introduced into the wavefront
modulating section 107, the wavefront of the laser light is
modulated according to the phase pattern displayed on the wavefront
modulating device 112. The modulated laser light passes through the
relay lenses 108, the scanner 109, and the relay lenses 110, is
reflected by the dichroic mirror 112, and enters the entrance pupil
of the objective lens 113 (step S105).
[0127] Because the entrance pupil position of the objective lens
113 is located at a position that is optically conjugate with the
wavefront modulating device 112, the laser light entering the
entrance pupil position has a wavefront identical to the wavefront
at the instant it is modulated by the wavefront modulating device
112.
[0128] Then, by focusing this laser light with the objective lens
113, a hologram image like one in which light is focused
simultaneously at a plurality of focal points located at different
positions can be projected inside the specimen A.
[0129] By simultaneously irradiating a plurality of specified focal
points with the laser light, in the state where a stimulus is
applied to the specimen A, the fluorescence emitted from the
specimen A is collected by the objective lens 113, is transmitted
through the dichroic mirror 121, is reflected by the mirror 117, is
focused by the focusing lens 120, and is acquired by the camera
116. Thus, it is possible to obtain a fluorescence image of the
specimen A while a light stimulus is applied thereto.
[0130] Thus, with the hologram-image projection apparatus 101 and
the hologram-image projection method according to this embodiment,
it is possible to focus laser light simultaneously at a plurality
of sites of interest located at different positions in the specimen
A. Consequently, when the focal points are set at positions
responding to a light stimulus in the specimen A, an advantage is
afforded in that it is possible to correctly observe the behavior
of the specimen A occurring directly after the light stimulus
without any time delay.
[0131] Note that, in this embodiment, although wavefront combining
is performed in the wavefront combining section 125 by calculating
a linear summation of the wavefronts, instead of this, it may be
performed by arraying divided regions of the plurality of
wavefronts calculated for individual focal points with a ratio
equal to the reciprocal of the total number of focal points.
[0132] More specifically, if n is the total number of focal points,
a linear summation of the wavefronts calculated for the individual
focal points is not calculated, but the region on the wavefront
modulating device 112 is divided, and any of the plurality of focal
points are associated with the individual divided regions. The
associating process here is preferably performed so that the
regions associated with the individual focal points are
periodically distributed with a ratio equal to the reciprocal of
the total number of focal points, n. Also, the distribution of the
regions associated with the individual focal points may be in the
form of a mosaic or may be in form of concentric circles. Assuming
this kind of association, in the wavefronts of the individual focal
points which are calculated in advance, wavefront elements of
portions of the wavefront modulating device 112 overlapping the
associated regions are extracted, and the wavefronts are combined
by arraying the extracted wavefront elements over the entire
region. The phase pattern to be applied to the wavefront modulating
device 112 is set on the basis of the combined wavefront obtained
in this way.
[0133] With the hologram-image projection method of this
embodiment, once the position information of each of the focal
points located at different positions in the specimen A is set in
the position setting step S101, in the wavefront calculating step
S102, reverse ray tracing is performed from each focal point to the
entrance pupil position of the objective lens 113, and a plurality
of wavefronts at the entrance pupil position of the laser light are
calculated from the individual focal points. By combining the
plurality of calculated wavefronts in the wavefront combining step
S103, a combined wavefront serving as the wavefront required to be
incident at the entrance pupil position of the objective lens to
simultaneously focus the laser light at different positions on the
focal plane is obtained by calculation. Then, a phase pattern is
applied to the wavefront modulating device 112 so that the
wavefront of the laser light incident on the wavefront modulating
device 112 is modulated to a wavefront identical to the obtained
combined wavefront.
[0134] In this case, because reverse ray tracing is performed by
using the lens data of the individual lenses constituting the
objective lens 113 in addition to the position information of the
individual focal points and the refractive index of the specimen,
the calculated wavefront contains the effects of aberrations
present in the objective lens 113. Therefore, by causing the laser
light having this wavefront to be incident on the entrance pupil
position of the objective lens 113 from the reflection side of the
specimen, the laser light can be focused on the focal plane of the
objective lens 113 with superior precision. Accordingly, by making
the laser light incident on the wavefront modulating device 112
which is given a phase pattern that yields such a wavefront, a
hologram image like one in which the laser light is focused
simultaneously at a plurality of focal points on the focal plane of
the objective lens 113 can be projected inside the specimen A.
[0135] With the hologram-image projection apparatus 101 of this
embodiment, when the position information of the individual focal
points located at different positions in the specimen A is set via
the input section 123, reverse ray tracing from the focal points to
the entrance pupil position of the objective lens 113 is performed
by the wavefront calculating section 124, and a plurality of
wavefronts at the entrance pupil position of the laser light are
calculated from the individual focal points. By combining the
plurality of calculated wavefronts with the wavefront combining
section 125, a combined wavefront serving as the wavefront required
to be incident at the entrance pupil position of the objective lens
113 to simultaneously focus the laser light at different positions
on the focal plane is obtained by calculation. Then, the phase
pattern is set by the phase-pattern setting section 126 on the
basis of the combined wavefront obtained in this way.
[0136] In this case, because reverse ray tracing is performed by
using the lens data of the individual lenses constituting the
objective lens 113 in addition to the position information of the
individual focal points and the refractive index of the specimen,
the calculated wavefront contains the effects of aberrations
present in the objective lens 113. Therefore, by causing laser
light having this wavefront to be incident at the entrance pupil
position of the objective lens from the reflection side of the
specimen, the laser light can be focused on the focal plane of the
objective lens with superior precision. Accordingly, by making the
laser light incident on the wavefront modulating device 112 when a
phase pattern that yields such a wavefront is applied to the
wavefront modulating device 112, a hologram image like one in which
laser light is focused simultaneously at a plurality of focal
points on the focal plane of the objective lens 113 can be
projected inside the specimen.
[0137] A hologram-image projection apparatus and hologram-image
projection method according to a fourth embodiment of the present
invention will be described below with reference to FIGS. 10, 11,
12A, and 12B.
[0138] In the third embodiment, the wavefront is created by
performing reverse ray tracing from each of the set focal points to
the entrance pupil position of the objective lens 113. In this
embodiment, however, instead of this, a spot image is obtained by
performing forward ray tracing from the entrance pupil position of
the objective lens to each focal point, and the wavefront is
created by minimizing the diameter of this spot.
[0139] As shown in FIG. 10, a hologram-image projection apparatus
200 according to this embodiment differs from the third embodiment
in the configuration of a control apparatus 104. Specifically, the
control apparatus 104 includes a storage section 122 that stores
the lens data of the individual lenses constituting an objective
lens 113 and the refractive index of a specimen A; an input section
(position-information setting section) 123 for setting position
information of the focal points of the laser light in the specimen
A; a virtual-image creating section 127 that creates a virtual
image having a plurality of spots formed at respective focal points
by performing forward ray tracing from the entrance pupil position
of the objective lens to the focal plane of the objective lens on
the basis of the lens data of each constituent lens of the
objective lens 113 and the refractive index of the specimen A
stored in the storage section 122; a wavefront calculating section
128 that calculates the laser light wavefront at the entrance pupil
position of the objective lens so that the diameter of the spots in
the created virtual image is minimized; and a phase-pattern setting
section 126 that sets the phase pattern to be applied to a
wavefront modulating device 112 on the basis of the wavefront
calculated by the wavefront calculating section 128.
[0140] Here, the forward ray tracing in the virtual-image creating
section 127 is performed using the lens data of the individual
constituent lenses of the objective lens 113 and the refractive
index of the specimen A.
[0141] Also, the wavefront calculating section 128 performs
calculations to vary the wavefront of the laser light at the
entrance pupil position of the objective lens and calculates the
diameters of the spots in the virtual image by forward ray tracing.
Then, by repeating this operation, the wavefront calculating
section 128 calculates the laser light wavefront that minimizes the
diameters of the spots in the created virtual image.
[0142] A hologram-image projection method for projecting a hologram
image like one in which laser light is focused simultaneously at a
plurality of different positions in the specimen A by using the
thus-configured hologram-image projection apparatus 200 according
to this embodiment will be described.
[0143] First, as shown in FIG. 11, position information for each
focal point on the focal plane of the objective lens is set via the
input section 123 (step S111). When the focal points are set to
positions that respond to a light stimulus in the specimen A, this
is performed by specifying individual focal points in the slice
image acquired at the focal plane of the objective lens via the
input section 123.
[0144] Next, if it assumed that the objective lens 113 is
aberration-free, a focal point map in which all focal points are
focused at different positions on the focal plane of the objective
lens 113 to form spots R is created in the input section 123 (step
S112).
[0145] Incidentally, because aberrations actually exist in the
objective lens 113, even if laser light for obtaining a hologram
image like that shown in FIG. 12A is made incident by the
aberration-free objective lens 113, in reality, one spot R1 is
distorted due to aberrations and becomes larger, as shown in FIG.
12B. Thus, in the virtual-image creating section 127, the focal
point map is Fourier transformed to calculate the wavefront of the
laser light at the entrance pupil position of the objective lens
113 (step S113). Then, for laser light having the calculated
wavefront at the entrance pupil position, forward ray tracing from
the entrance pupil position of the objective lens 113 to the focal
plane is performed in the virtual-image creating section 127 using
the lens data of each lens constituting the objective lens 113 and
the refractive index of the specimen A, to create the virtual image
(step S114). During this process, the influence of the actual
aberrations is reflected in the created virtual image, and the spot
R1 is distorted due to the aberrations and becomes larger.
[0146] For each focal point forming the spot R1, the wavefront, at
the entrance pupil position of the objective lens 113, of the laser
light entering the objective lens 113 is repeatedly modified, and
the wavefront that minimizes the spot diameter of the spot R1 is
calculated (step S115). More specifically, as a metric for
evaluating the spot diameter of each spot R1, Zernike polynomial
coefficients are repeatedly varied, and the Zernike polynomial
coefficients that minimize the spot diameter, serving as the
evaluation metric, are identified.
[0147] Here, a cylindrical function w(r, .theta.) that gives the
shape of the wavefront, which is a Zernike polynomial, is shown in
the following expression:
w ( r , .theta. ) = Z 1 + Z 2 .rho.cos .theta. + Z 3
.rho.sin.theta. + Z 4 ( 2 .rho. 2 - 1 ) + Z 5 .rho. 2 cos 2 .theta.
+ Z 6 .rho. 2 sin 2 .theta. + Z 7 ( 3 .rho. 2 - 2 ) .rho. cos
.theta. + Z 8 ( 3 .rho. 2 - 2 ) .rho.sin .theta. ##EQU00002##
Here, .rho. is the distance from the center of the pupil normalized
by the radius from the center of the entrance pupil of the
objective lens 113, and .theta. is the angle relative to a
prescribed reference line in polar coordinates in the plane of the
wavefront modulating device 112 or the entrance pupil plane of the
objective lens 113. Zn are Zernike coefficients.
[0148] The wavefront calculated in this way is input to the
phase-pattern setting section 126, and the phase pattern to be
applied to the wavefront modulating device 112 is set to be
identical to the wavefront at the entrance pupil position of the
objective lens 113 or a pattern formed by phase-wrapping processing
(step S116). Then, the phase pattern set by the phase-pattern
setting section 126 is output to the wavefront modulating device
112. In this state, when the laser light is emitted from the laser
light source 105 and is introduced to the wavefront modulating
section 7, the wavefront of the laser light is modulated according
to the phase pattern on the wavefront modulating device 112. The
modulated laser light passes through the relay lenses 108, the
scanner 109, the relay lenses 110, and the dichroic mirror 121 and
is focused onto the specimen by the objective lens 113 to irradiate
it (step S117).
[0149] By doing so, because forward ray tracing is performed in the
virtual-image creating section 127 by using the lens data of the
individual lenses constituting the objective lens 113 in addition
to the position information of the individual focal points and the
refractive index of the specimen A, the spots in the created
virtual image contain the influence of the aberrations existing in
the objective lens 113. Therefore, by varying the Zernike
polynomial coefficients so as to minimize the spot diameter of the
spot R1 in which deformation occurs due to the aberrations, the
wavefront at the entrance pupil position of the objective lens 113
is modulated to a wavefront in which the aberrations are
anticipated in advance.
[0150] Therefore, by making laser light having such a wavefront
incident at the entrance pupil position of the objective lens 113
from the opposite side of the specimen A, the wavefront is restored
while being focused by the objective lens 113 and can be focused on
the focal plane with superior precision. Therefore, by making the
laser light incident on the wavefront modulating device 112 which
has been given a phase pattern that yields such a wavefront, a
hologram image like one in which laser light is focused
simultaneously at a plurality of focal points on the focal plane of
the objective lens 113 can be projected in the specimen A.
[0151] In this embodiment, although a segmented MEMS mirror whose
surface shape can be varied has been illustrated as an example of
the wavefront modulating device 112, instead of this, any other
type of wavefront modulating device 112 can be used, for example, a
liquid crystal device, a deformable mirror, or the like. In the
case of a liquid crystal device, the refractive index distribution
due to the alignment of the liquid crystal molecules constitutes
the phase pattern, and in the case of a deformable mirror, the
surface shape thereof constitutes the phase pattern.
[0152] With the hologram-image projection method of this
embodiment, once the position information of the individual focal
points disposed at different positions in the specimen is set in
the position setting step S111, in the virtual-image creating step
S114, forward ray tracing is performed from the entrance pupil
position of the objective lens to the focal plane, and a virtual
image having a plurality of spots formed at the individual focal
points is created. Then, in the wavefront calculating step S115,
the laser light wavefront at the entrance pupil position of the
objective lens 113 is calculated so that the diameters of the
individual spots in the created virtual image are minimized. Then,
a phase pattern is applied to the wavefront modulating device 112
on the basis of the wavefront calculated in the wavefront
calculating step S115 so that the wavefront of the laser light
incident on the wavefront modulating device 112 is modulated to a
wavefront identical to the obtained wavefront.
[0153] In this case, because the forward ray tracing is performed
using the lens data of the individual lenses constituting the
objective lens 113, in addition to the position information of each
focal point and the refractive index of the specimen, the
individual spots in the created virtual image contain the influence
of the aberrations present in the objective lens 113. Therefore, by
making laser light having a wavefront that minimizes the diameters
of the individual spots incident at the entrance pupil position of
the objective lens 113 from the opposite site of the specimen, the
laser light is focused on the focal plane of the objective lens 113
with superior precision. Therefore, by introducing laser light to
the wavefront modulating device 112 which is given a phase pattern
that yields such a wavefront, a hologram image like one in which
laser light is focused simultaneously at a plurality of focal
points on the focal plane of the objective lens 113 can be
projected inside the specimen.
[0154] A hologram-image projection apparatus according to a fifth
embodiment of the present invention will be described below with
reference to the drawings.
[0155] As show in FIG. 13, a hologram-image projection apparatus
201 according to this embodiment, which is a microscope system,
includes a light source apparatus 202 that emits laser light, a
microscope apparatus 203 that irradiates a specimen A with the
laser light incident from the light source apparatus 202, and a
control apparatus 204 that controls the laser light incident on the
microscope apparatus 203 from the light source apparatus 202.
[0156] The light source apparatus 202 includes a laser light source
205 that emits laser light, a collimator lens 206 that converts the
laser light emitted from the laser light source 205 into a
collimated beam, a wavefront modulating section 207 that modulates
the wavefront of the collimated laser light, relay lenses 208 and
210, and a scanner (incident-angle adjusting section) 209 that
scans the laser light.
[0157] The wavefront modulating section 207 includes a prism 211
that reflects the laser light and a reflective wavefront modulating
device 212 that reflects the laser light reflected by the prism
211, during which time the wavefront of the laser light is
modulated by a phase pattern, and returns the modulated laser light
to the prism 211.
[0158] The laser light reflected by the prism 211 has its light
path folded at the wavefront modulating device 212 so as to return
to the same prism 211, and then returns to a light path on the same
axis as the laser light from the laser light source 205.
[0159] The wavefront modulating device 212 is formed of a segmented
MEMS mirror whose surface shape can be arbitrarily changed by the
control apparatus 204, described later. In this case, the surface
shape formed by the indentations and protrusions of the individual
segments of the MEMS mirror constitutes the phase pattern for
modulating the wavefront of the laser light. The wavefront
modulating device 212 and the entrance pupil position of an
objective lens 213 are disposed in an optically conjugate
positional relationship.
[0160] The scanner 209 is a so-called proximity galvanometer mirror
in which two galvanometer mirrors 209a and 209b that can be
swiveled about axes disposed in mutually intersecting directions
are placed in close proximity to each other, so that the incident
laser light can be scanned two-dimensionally.
[0161] The microscope apparatus 203 includes the objective lens
213, which focuses the laser light onto the specimen A, disposed on
a stage 214, and also collects light coming from the specimen A; a
light detector 215, formed of a photomultiplier tube, for detecting
the light collected by the objective lens 213; a camera 216, such
as a CCD, that captures a fluorescence image in the specimen A; and
a mirror 217 that is inserted in and removed from the light path so
as to switch the light path to either the light detector 215 or the
camera 216. Reference numerals 218 to 220 are focusing lenses, and
reference numeral 221 is a dichroic mirror. The objective lens 213
is provided in such a manner that the distance between the
objective lens 213 and the stage 214 in the optical axis direction
can be varied.
[0162] By setting the surface shape of the wavefront modulating
device 212 to a flat reflective surface shape, it is possible to
make laser light having a plane-wave wavefront incident at the
entrance pupil position of the objective lens 213. Accordingly, it
is possible to focus the laser light at the focal plane of the
objective lens 213.
[0163] With the mirror 217 switched to the light detector 215 side
(a position where it is removed from the light path, as indicated
in the dotted lines), the laser light is emitted from the laser
light source 205, and while the laser light focused at the focal
plane in the specimen A is two-dimensionally scanned by driving the
scanner 209, fluorescence produced at each focal position is
detected by the light detector 215, thereby enabling acquisition of
a two-dimensional fluorescence image of the specimen A extending
over the focal plane of the objective lens 213.
[0164] Then, by acquiring a plurality of two-dimensional
fluorescence images (slice images) while changing the position of
the focal plane of the objective lens 213 by changing the relative
distance between the objective lens 213 and the stage 214, it is
possible to acquire a three-dimensional fluorescence image of the
specimen A.
[0165] As shown in FIG. 13, the control apparatus 204 includes an
input section (focal position setting section) 222 for setting
position information of the laser light at the specimen A; a
control unit 224 that controls the scanner 209 and the
optical-path-length adjusting prism 223, described later, on the
basis of the position information of the individual focal points
set via the input section 222; a wavefront setting section 225 that
measures the wavefront of return light from the individual focal
points at positions set via the input section 222; a wavefront
combining section 226 that combines a plurality of wavefronts of
the return light, measured for all focal points; and a
phase-pattern setting section 227 that sets a phase pattern to be
applied to the wavefront modulating device 212 on the basis of the
combined wavefront combined by the wavefront combining section
226.
[0166] The input section 222 sets the position information of each
focal point by the observer specifying the positions where the
laser light is desired to be focused, that is to say, the focal
points, on a monitor (not illustrated).
[0167] Here, the focal points on the specimen A may be set to
arbitrary positions, or they may be set to position that respond to
a light stimulus. When the focal points are desired to be set to
positions that respond to a light stimulus, it is preferable to
display the image acquired by the microscope apparatus 203 on the
monitor (not illustrated) so as to make is easier for the observer
to specify the focal points.
[0168] The wavefront measuring section 225 includes a polarizing
beam splitter (splitting section) 228, disposed in front of the
wavefront modulating section 207, for splitting the laser light
into reference light and measurement light; a polarizing beam
splitter 231, disposed after the wavefront modulating section 207
which is provided in a measurement light path 229 along which the
measurement light travels, for combining the returning measurement
light from the specimen A and the reference light coming via the
reference light path 230; a wave plate 232 that rotates the
polarization direction of the laser light, which is converted to a
collimated beam by the collimator lens 206, by an arbitrary angle;
a wave plate 233 that converts the laser light (measurement light)
transmitted through the polarizing beam splitter 231 into
circularly polarized light or rotates it by 45.degree.; and a
detection light path 234 for detecting the reference light and the
return light combined by the polarizing beam splitter 231.
[0169] The wave plate 232 is placed so as to rotate the
polarization direction of the laser light, so that the laser light
is split at the polarizing beam splitter 228 into the reference
light and the measurement light at a prescribed intensity ratio.
The wave plate 233 is disposed so as to rotate the polarization
direction by 90.degree. in the section where the measurement light
transmitted through the polarizing beam splitter 231 is focused at
the specimen A and then return light from the specimen A re-enters
the polarizing beam splitter 231.
[0170] An optical-path-length adjusting prism 223 that is provided
so as to be movable along the optical axis to adjust the optical
path length, a dispersion-compensation plate 235 that compensates
for group velocity dispersion, and a half-wave plate 236 that
rotates the polarization direction of the reference light entering
the polarizing beam splitter 231 by 90.degree. are disposed in the
reference light path 230. Reference numeral 237 is a mirror.
[0171] A polarizing plate 238 that transmits the return light
passing through the wave plate 233 and the reference light passing
through the half-wave plate 236 with a prescribed intensity ratio;
relay lenses 239 that relay the pupil; and an interference-light
detector 240 that detects the interference light generated by
combining the return light and the reference light are disposed in
the detection light path 234.
[0172] Because the polarization directions of the return light
passing through the wave plate 233 and the reference light passing
through the half-wave plate 236 are substantially orthogonal to
each other, the polarizing plate 238 has a transmission axis
forming an angle greater than 0 relative to the polarization
directions of the respective beams. Accordingly, the polarizing
plate 238 transmits only components of the return light and the
reference light oriented along a prescribed axis.
[0173] The interference-light detector 240 is disposed so as to
have an optically conjugate positional relationship with the
wavefront modulating device 212 and the entrance pupil position of
the objective lens 213.
[0174] The wavefront combining section 226 is configured to combine
the wavefronts measured for all of the focal points. In this
embodiment, the wavefront combining section 226 is configured to
calculate the linear summation of the wavefronts calculated for all
of the focal points.
[0175] The phase-pattern setting section 227 sets the phase pattern
to be applied to the wavefront modulating device 212 on the basis
of the combined wavefront calculated by the wavefront combining
section 226 and outputs it to the wavefront modulating device 212.
Here, because the wavefront modulating device 212 has a conjugate
relationship with the entrance pupil of the objective lens 213, the
phase pattern applied to the wavefront modulating device 212 is
identical to the combined wavefront at the entrance pupil position
of the objective lens 213 or a pattern formed by phase-wrapping
processing. In phase-wrapping processing, when the range of phase
modulation at the wavefront modulating device 212 is set to 2 n.pi.
(n is an integer), for portions in the combined wavefront at the
entrance pupil position of the objective lens 213 that have a phase
difference exceeding 2 n.pi., 2 n.pi. is subtracted from that phase
difference. With the segmented MEMS mirror in this embodiment,
because the range of phase modulation is normally set to a range of
2 n.pi., phase-wrapping processing is performed as required.
[0176] In this way, the wavefront modulating device 212 is adjusted
to a shape matching the input phase pattern. Thus, the laser light
reflected at the wavefront modulating device 212 is modulated at
the surface of the wavefront modulating device 212 to have the same
wavefront as the combined wavefront obtained at the entrance pupil
position of the objective lens 213. Therefore, by focusing this
laser light with the objective lens 213, it is simultaneously focus
it at the individual set focal points while keeping the objective
lens 213 fixed.
[0177] The operation of the thus-configured hologram-image
projection apparatus 201 according to this embodiment will be
described below.
[0178] First, the position information of the individual focal
points is set via the input section 222.
[0179] Here, when the focal point is set to a position in the
specimen A that responds to a light stimulus, the wavefront
modulating device 212 is set to a phase pattern that gives a flat
reflecting surface shape, based on the output from the control
apparatus 204. In the microscope apparatus 203, the mirror 217 is
retracted from the light path. Then, laser light is emitted from
the laser light source 205, and the laser light is
two-dimensionally scanned by the scanner 209.
[0180] The laser light emitted from the laser light source 205
propagates along the light path without changing its wavefront and,
after being two-dimensionally scanned by the scanner 209, is
reflected by the dichroic mirror 221 to enter the objective lens
213 and is focused at the focal plane in the specimen A.
Fluorescence is produced at the focal position of the laser light,
and the fluorescence produced is collected by the objective lens
213, is transmitted through the dichroic mirror 221, is focused by
the focusing lenses 219 and 220, and is detected by the light
detector 215.
[0181] Because the fluorescence is produced only in an extremely
shallow region in the vicinity of the focal plane of the objective
lens 213, by storing the intensity of the fluorescence detected by
the light detector 215 and the scanning position of the laser light
scanned by the scanner 209 in association with each other, it is
possible to obtain a two-dimensional fluorescence image (slice
image) of the specimen A extending over the focal plane.
[0182] Also, a three-dimensional fluorescence image can be obtained
by acquiring a plurality of slice images while moving the objective
lens 213 and the specimen A relative to each other in the optical
axis direction.
[0183] On the two-dimensional or three-dimensional fluorescence
image displayed on the monitor (not shown), the observer specifies
the position of focal points where the laser light is desired to be
focused. For example, if the specimen A is a nerve cell whose
behavior is to be observed when stimulated with laser light, the
focal points where the stimulus is to be applied are distributed
three-dimensionally. In this case, the observer sets the
three-dimensional position information of the focal points by
specifying all focal points.
[0184] Next, wavefront measurement is performed in the wavefront
measuring section 225 for the individual set focal points.
[0185] Specifically, the control unit 224 operates the scanner 209
on the basis of the position coordinates of each of the focal
points set via the input section and irradiates the focal points
with laser light one-by-one. During this process, the control unit
224 adjusts the position of the optical-path-length adjusting prism
223 so that the optical path length of the reference light path 230
matches the optical path length of the measurement light path 229
to the individual focal points set via the input section 222. Also,
during this process, the wavefront modulating device 212 is set to
a phase pattern giving a flat reflective surface shape.
[0186] Laser light having, for example, a vertical polarization
plane emitted from the laser light source 205 is made to pass
through the wave plate 232, thereby rotating the polarization
direction thereof by a prescribed angle, and then enters the
polarizing beam splitter 228. At the polarizing beam splitter 228,
the light is split into two, a vertically polarized component and a
horizontally polarized component, one of which, for example, the
vertically polarized component, is introduced to the reference
light path 230, and the other of which is introduced to the
measurement light path 229.
[0187] The vertically polarized component directed to the reference
light path 230 is subjected to dispersion compensation upon passing
through the dispersion-compensation plate 235, and after being
folded at the optical-path-length adjusting prism 223, the
polarization direction is rotated by 90.degree. by the half-wave
plate 236 to become a horizontally polarized component. The laser
light from the reference light path 230, which has become the
horizontally polarized component, is transmitted through the
polarizing beam splitter 231 and is introduced to the detection
light path 234.
[0188] On the other hand, the horizontally polarized component
transmitted through the polarizing beam splitter 228 enters the
measurement light path 229 and, after being reflected at the prism
211 and the wavefront modulating device 212, is transmitted through
the polarizing beam splitter 231 and passes through the wave plate
233. Accordingly, the laser light that has been converted to
circularly polarized light or had its polarization direction
rotated by 45.degree. passes through the relay lenses 208 and is
then given an angle for directing it to a desired focal point by
the scanner 209. Then, after being transmitted through the relay
lenses 210, the light enters the microscope apparatus 203.
[0189] The laser light that has entered the microscope apparatus
203 is reflected by the dichroic mirror 221 and is focused at the
specimen A by the objective lens 213. The laser light reflected at
a region in the vicinity of the focal point in the specimen A is
collected by the objective lens 213, and is then reflected by the
dichroic mirror 221, returns via the relay lenses 210, the scanner
209, and the relay lenses 208, is converted to a vertically
polarized component by the wave plate 233, and enters the
polarizing beam splitter 231.
[0190] The laser light formed of the vertically polarized component
entering the polarizing beam splitter 231 is reflected by the
polarizing beam splitter 231 and enters the detection light path
234. At this time, the laser light formed of the vertically
polarized component is combined with the laser light formed of the
horizontally polarized component coming via the reference light
path 230. Then, in the laser light formed of the vertically
polarized component, which is the return light from the specimen A,
and the laser light formed of the horizontally polarized component,
which is the reference light, only the components parallel to the
transmission axis of the polarizing plate 238 are transmitted
through the polarizing plate 238 and are incident on the
interference-light detector 240 via the relay lenses 239. Because
the return light and the reference light transmitted through the
polarizing plate 238 have the same polarization direction,
interference between the return light and the reference light is
possible. Also, because the optical path length of the reference
light path 230 and the optical path length of the measurement light
path 229 up to the focal point are made the same by the
optical-path-length adjusting prism 223, only the return light
returning from the focal point interferes with the reference
light.
[0191] Accordingly, the differences between the wavefront of the
laser light emitted from the laser light source 205 and the
wavefront of the laser light which is return light from the focal
point are detected at the interference-light detector 240 as an
interference pattern.
[0192] Once the wavefronts are detected by performing the
above-described operation for all focal points, the linear
summation of the wavefronts measured for the individual focal
points is calculated by the wavefront combining section 226, and
the combined wavefront is created. The created combined wavefront
is input to the phase-pattern setting section 227, and the phase
pattern to be applied to the wavefront modulating device 212 is set
so as to be identical to the combined wavefront at the entrance
pupil position of the objective lens or a pattern obtained by
phase-wrapping processing. Then, the phase pattern set by the
phase-pattern setting section 227 is output to the wavefront
modulating device 212.
[0193] This completes the preparations for observing the specimen A
while performing optical stimulation.
[0194] In this state, the microscope apparatus 203 is configured so
that the mirror 217 is inserted into the light path (disposed at a
position indicated by the solid lines in the figure), and the
fluorescence collected by the objective lens 213 is acquired by the
camera 216. Then, with the scanner 9 stopped at the origin, when
the laser light emitted from the laser light source 205 is
introduced into the wavefront modulating section 207, the wavefront
of the laser light is modulated according to the phase pattern
displayed on the wavefront modulating device 212. The modulated
laser light passes through the relay lenses 208, the scanner 209,
and the relay lenses 210, is reflected by the dichroic mirror 221,
and enters the entrance pupil of the objective lens 213.
[0195] Because the entrance pupil position of the objective lens
213 is located at a position that is optically conjugate with the
wavefront modulating device 212, the laser light incident at the
entrance pupil position has a wavefront identical to the wavefront
at the instant it is modulated by the wavefront modulating device
212.
[0196] Then, by focusing this laser light with the objective lens
213, a three-dimensional hologram image like one in which light is
focused simultaneously at a plurality of focal points located at
different positions can be projected inside the specimen A.
[0197] By simultaneously irradiating a plurality of specified focal
points with the laser light, in the state where a stimulus is
applied to the specimen A, the fluorescence emitted from the
specimen A is collected by the objective lens 213, is transmitted
through the dichroic mirror 221, is reflected by the mirror 217, is
focused by the focusing lens 218, and is acquired by the camera
216. Thus, it is possible to obtain a fluorescence image of the
specimen A while a light stimulus is applied thereto.
[0198] Thus, with the hologram-image projection apparatus 201
according to this embodiment, because the wavefront of the laser
light is adjusted so that the combined wavefront formed by
combining the wavefronts obtained by measuring the return light
from the point light sources disposed at all focal points is
incident at the entrance pupil position of the objective lens 213,
it is possible to focus the laser light at a plurality of focal
points simultaneously with superior precision to apply a light
stimulus to the specimen A. As a result, when the focal points are
set at positions in the specimen that respond to a light stimulus,
an advantage is afforded in that it is possible to correctly
observe the behavior of the specimen A occurring directly after the
light stimulus without any time delay.
[0199] In this case, according to this embodiment, because the
wavefronts corresponding to the individual focal points are
obtained by measurement, even in the presence of various
aberrations whose cause cannot be ascertained, an advantage is
afforded in that they can be sufficiently compensated for without
identifying their cause, and the laser light can be focused with
superior precision at the individual focal points.
[0200] In this embodiment, a device of the type in which the laser
light is divided into reference light and measurement light, the
reference light and the return light are made to interfere by
making the optical path length of the measurement light path 229
match that of the reference light path 230, and the wavefront is
measured is employed as the wavefront measuring section 225;
however, as shown in FIG. 14, a device of the type in which a
confocal pinhole (pinhole member) 241 and a Hartmann sensor 242 are
combined may be used instead.
[0201] That is, in the example shown in FIG. 14, the polarizing
beam splitter 228 and the reference light path 230 are eliminated,
and instead, the confocal pinhole is disposed in the detection
light path 234, and a 2 Hartmann sensor 242 is employed instead of
the interference-light detector 240. By disposing the confocal
pinhole at a position that is optically conjugate with the focal
position of the objective lens 213, it is possible to detect, with
the Hartmann sensor 242, only the return light from a point light
source disposed at the focal position and to measure the
wavefront.
[0202] In this figure, the wave plate 232 is an element for
rotating the polarization plane so that the polarization direction
of the laser light emitted from the laser light source 205 matches
the polarization direction that can be transmitted through the
polarizing beam splitter 231.
[0203] In this embodiment, the wavefront combining in the wavefront
combining section 226 is performed by calculating the linear
summation of the wavefronts; instead of this, however, it may be
performed by arraying divided regions of the plurality of
wavefronts calculated for the individual focal points with a ratio
equal to the reciprocal of the total number of focal points.
[0204] More specifically, if n is the total number of focal points,
a linear summation of the wavefronts calculated for the individual
focal points is not calculated, but the region on the wavefront
modulating device 212 is divided, and any of the plurality of focal
points are associated with the individual divided regions. The
associating process here is preferably performed so that the
regions associated with the individual focal points are
periodically distributed with a ratio equal to the reciprocal of
the total number of focal points, n. Also, the distribution of the
regions associated with the individual focal points may be in the
form of a mosaic or may be in form of concentric circles. Assuming
this kind of association, in the wavefronts from the individual
focal points which are calculated in advance, wavefront elements of
portions of the wavefront modulating device 212 overlapping the
associated regions are extracted, and the wavefronts are combined
by arraying the extracted wavefront elements over the entire
region. The phase pattern to be applied to the wavefront modulating
device 212 is set on the basis of the combined wavefront obtained
in this way.
[0205] Furthermore, in this embodiment, although a segmented MEMS
mirror whose surface shape can be varied has been illustrated as an
example of the wavefront modulating device 212, instead of this,
any other type of wavefront modulating device 212 can be used, for
example, a liquid crystal device, a deformable mirror, or the like.
In the case of a liquid crystal device, the refractive index
distribution due to the alignment of the liquid crystal molecules
constitutes the phase pattern, and in the case of a deformable
mirror, the surface shape thereof constitutes the phase
pattern.
[0206] With the hologram-image projection apparatus 201 of this
embodiment, when the laser light emitted from the laser light
source is radiated onto the specimen A, the wavefronts, at the
entrance pupil position, of the return light returning from the
individual focal points at the positions set via the input section
222 are measured by the wavefront measuring section 225. Once the
wavefronts are measured, the wavefront combining section 226
calculates the combined wavefront by combining the plurality of
measured wavefronts corresponding to the individual focal points,
and the phase-pattern setting section 227 sets the phase pattern to
be applied to the wavefront modulating device 212 on the basis of
the calculated combined wavefront and outputs this phase pattern to
the wavefront modulating device 212. Then, by making laser light
incident on the wavefront modulating device 212 with the set phase
pattern applied to the wavefront modulating device 212, laser light
having the combined wavefront is made incident at the entrance
pupil position of the objective lens 213, and a hologram image
including a plurality of focal points is projected inside the
specimen A.
[0207] In this case, with the hologram-image projection apparatus
201 according to this embodiment, because the phase pattern for
focusing the laser light at the focal points is set by measuring
the wavefronts of return light from a plurality of focal points
that actually exist, even when causes of various aberrations
coexist, the laser light can be focused simultaneously at a
plurality of desired focal points in the specimen.
[0208] According to this embodiment, by splitting the laser light
with the polarizing beam splitter 228, one laser beam is directed
to the specimen to irradiate the specimen, while the other laser
beam serves as reference light. Then, the reference light and the
return light from point light sources disposed at focal points set
in the specimen A are made to interfere by combining them with the
polarizing beam splitter 231, and the interference light thereof is
detected by the interference-light detector 240. Accordingly,
because minute differences in the wavefronts of the reference light
and the return light from the specimen are detected by the
interference-light detector 240, the laser light wavefront for
focusing the laser light at the focal point can be precisely
measured as the differences relative to the reference light.
[0209] Also, of the return light returning from the specimen as a
result of irradiating the specimen with laser light, only the
return light emitted from the focal position of the objective lens
is selected by the confocal pinhole 241 and can be detected by the
Hartmann sensor 242. Thus, it is possible to accurately measure the
wavefront of the return light from a point light source disposed at
the focal position of the objective lens 213.
REFERENCE SIGNS LIST
[0210] A specimen [0211] .DELTA.d prescribed distance [0212] P
focal point [0213] Q reference plane [0214] R, R1, R2 spot [0215]
.DELTA.Z change in vector information [0216] S1, S11 step of
setting position information [0217] S2 step of calculating laser
light wavefront at entrance pupil position [0218] S3 step of
calculating combined wavefront [0219] S4, S16 step of setting phase
pattern [0220] S5 step of focusing laser light a specimen [0221]
S12 step of creating first virtual image [0222] S14 step of
creating second virtual image [0223] 1, 100 hologram-image
projection apparatus [0224] 12 wavefront modulating device [0225]
13 objective lens [0226] 23 input section (position-information
setting section) [0227] 24 wavefront calculating section [0228] 25
wavefront combining section [0229] 26 phase-pattern setting section
[0230] S101, S111 step of setting position [0231] S102, S115 step
of calculating wavefront at entrance pupil position [0232] S103
step of calculating combined wavefront [0233] S104, S116 step of
setting phase pattern [0234] S105, S117 step of focusing modulated
laser light at specimen [0235] S112 step of creating focal point
map [0236] S114 step of creating virtual image [0237] 101
hologram-image projection apparatus [0238] 112 wavefront modulating
device [0239] 113 objective lens [0240] 123 input section
(position-information setting section) [0241] 124 wavefront
calculating section [0242] 125 wavefront combining section [0243]
126 phase-pattern setting section [0244] 201 hologram-image
projection apparatus [0245] 205 laser light source [0246] 209
scanner (incident-angle adjusting section) [0247] 212 wavefront
modulating device [0248] 213 objective lens [0249] 222 input
section (focal-position setting section) [0250] 225 wavefront
measuring section [0251] 226 wavefront combining section [0252] 227
phase-pattern setting section [0253] 231 polarizing beam splitter
(combining section) [0254] 240 interference-light detector [0255]
241 confocal pinhole (pinhole member) [0256] 242 Hartmann
sensor
* * * * *